In Celebration of Larry Stein (1931 – 2019)
by Thomas A. Ban
On May 30, 2019, Larry Stein, a pioneer of behavioral pharmacology, passed away.
Larry was born November 10, 1931, and grew up in New York City. He graduated from Stuyvesant High School in 1949 and received his BA from New York University in 1952. Larry continued with his studies and earned his MA and PhD in Psychology, with distinction, from the University of Iowa in 1953 and 1955, respectively. In graduate school he was privileged to have Kenneth W. Spence, a distinguished learning theorist, as his mentor (Spence 1951).
After receiving his BA from New York University in 1952 he continued with his studies and earned his MA and PhD in Psychology, with distinction, from the University of Iowa in 1953 and 1955, respectively. In graduate school he was privileged to have Kenneth W. Spence, a distinguished learning theorist, as his mentor (Spence 1951).
A gifted teacher and dedicated researcher, Larry was instrumental in bridging behavioral pharmacology with neurochemistry by demonstrating the reward augmenting effect of dopamine agonists, such as apomorphineand high dose amphetamine (Ban 2011a; Baxter, Gluckman, Stein and Scerni 1974; Stein, Belluzzi, Ritter and Wise 1974).
In the heady days of the mid-1950s, as behavioral psychopharmacology was just opening-up, Larry Stein, at the Walter Reed Army Institute for Research (“the Reed”) in Washington, DC, sat at the very center of the action. There he had Joseph Brady, who was studying the effects of new dugs on emotional behavior in animals, as his mentor; a few miles away Bernard Brodie, at the Heart Institute of the National Institutes of Health (NIH), was just teasing apart the behavioral effects of the neurotransmitters; and the National Institute of Mental Health (NIMH) was at the halfway point in its decade-long transition from research on psychoanalysis to neurobiology (Brady 1956). With his work on amphetamine at “the Reed” and the Veterans Administration (VA) Hospital in Pittsburgh later in the 1950s – and at Wyeth Laboratories in the 1960s – Stein was instrumental in opening up the “catecholamine theory of brain stimulation – reinforcement“ (Shorter 2011a,b).
Reflecting on the story of his major contributions during his interview for the Oral History Series of the American College of Neuropsychopharmacology (ACNP), Larry shared with his distinguished interviewer, the Nobel Laureate Arvid Carlsson, a kind of eureka moment when he gave an account of the key observation to his discovery: “By God, I can still remember vividly Bruce Baxter (one of his co-workers at Wyeth Laboratories) coming up to me one day very excited with the tracings (of his polygraph) showing that rats loved to self-administer apomorphine!” Prior to this it was only known that amphetamine administration made rats vomit. So, that was a very big early hint that dopamine should get more prominence in reward theory.
Carlsson also asked him, rather puckishly, if he believed that “a single cell could have a mind?” Stein responded: “All this makes me a little bit uncomfortable because I was brought up in a very behavioristic tradition – to me, mind is almost a naughty word” (Shorter 2011a).
By 1971 Stein had moved on to the concept of schizophrenia representing “a reward system out of control” (Shorter 2011a; Stein 1962; Stein and Wise 1971). In 1979 Stein accepted the chair of Pharmacology at the University of California, Irvine; he became emeritus professor there in 2007 after his retirement.
The historic interview of Larry, with Carlsson as his interviewer, took place on December 13, 1995, in San Juan, Puerto Rico, at the annual meeting of the ANCP. The edited version, presented below, was first published in Volume One (Starting Up) of the series (Ban 2011b; Shorter 2011b; Stein 2011).
AC: I am Arvid Carlsson from the University of Gothenburg, Sweden, and I have the great pleasure to interview my old friend, Doctor Larry Stein.Doctor Stein is professor and chairman of the Department of Pharmacology, University of California at Irvine. The question I would like to start with Larry, is how you got into the area of central nervous system (CNS) pharmacology and which area has been most important for you?
LS: That is a frequent question, because my formal training was in psychology and behavior. the pharmacology was more informally acquired after I had been out in the field and had a job. my first contact with drugs and finding out how wonderfully drugs can be used to analyze behavior was when I was drafted into the Army after my doctoral work at Iowa was completed. I had the good fortune to be assigned to the Walter Reed Army Institute of Research in Washington, DC, and my mentor there was Joe Brady. they were doing some very new things at Walter Reed, two of which particularly stimulated my interest, both then and throughout the entirety of my career. The first was that Joe and others at the institute were working with the then-exciting new drugs, reserpine and chlorpromazine, and were starting to describe their behavioral effects. And secondly, Joe’s laboratory was the first to confirm the very important discovery of James Olds and Peter Milner that one could electrically stimulate the brain to produce reward effects. Specifically, Olds found that animals would work very hard to self-stimulate certain points in their own brains. Incidentally, it was Joe Brady who coined the term “self-stimulation” at that time.
AC: Yes, this concept of reward was new, entirely new at the time, wasn’t it? The whole idea that there is in the brain a system that operates to give us reward for what we are doing, wasn’t that entirely new?
LS: Yes, although it seems perfectly obvious now, since anything we can experience or feel is currently thought to be a result of brain activity. So it must be the case that if we can feel the reward, then the brain must be responsible for producing it. But the theories of positive reinforcement popular at that time were based in part on Freudian theory. Freud held the Victorian idea that reward is not so much excited by hedonically positive or pleasurable stimulation, but rather that it consists mainly in the reduction of pain, or satisfaction of drives such as hunger. Thus, most psychologists at that time favored drive-reduction theories of reward with the implication that reward was mediated by decreases in brain activity. According to these theories, it should have been impossible to electrically excite the brain and produce reward until James Olds’ discovery.
AC: One thing I would like to ask here. Olds, of course, discovered this, but did he really go into the enormous potential of this in terms of pharmacology?
LS: Yes, Olds did many of the early pharmacology studies and discovered, for example, that chlorpromazine had quite selective reward-diminishing effects at doses that did not significantly impair motor behavior. But he left a little segment of work for me. I will always appreciate Professor Olds for always using a little bit too high a dose when he studied amphetamine. He found that high doses of amphetamine, like all effective doses of chlorpromazine, will diminish reward effects. I was able to discover, by coming down in dose, that amphetamine’s very large reward-augmenting effects can readily be displayed. This finding opened up this little career of mine.
AC: That is an understatement. And also, I would guess that you must have been more or less the pioneer of the area of drug development using these techniques, or what do you say? I mean, that you are the one that really pioneered this, isn’t that true?
LS: I was one of the early workers and I think that it is fair to say that I was there at the time that we investigators were making up the field as we went along. But it would be taking too much credit to make me the pioneer of behavioral pharmacology. I would think that it was such people as Joe Brady and Peter Dews and a number of other investigators who first understood how nicely behavior and drugs go together. In contrast, electrophysiology and drugs didn’t quite seem to go as nicely together. It is almost as though drugs cut along the analytical lines of behavior in a very deep way. The chemical theory of neurotransmission was not so old at the time. But now, and in large part from your own efforts, Dr. Carlsson, we understand that messages are communicated in the brain by means of chemical transmitters. Thus, if one manipulates the brain’s chemical messaging system with drugs, it just makes very plausible sense that one can readily manipulate behavior.
AC: Yes, and this is how I understand your contributions in this area that you bring in the concept of chemical transmission into your thinking at the very, very early stages.
LS: Yes. I guess both of us were influenced by the early powerfully simplifying ideas of Bernard Brodie.
LS: Who dared to think that with as complicated an organ as the brain, the functions can be analyzed in terms of the actions and interactions of a small handful of neurotransmitters? This was just a wonderful and simplifying idea to be pushed as far as it would go. It is astonishing, I think, how far that simplifying idea can be pursued. Obviously in a complicated system such as the brain, everybody understands that it is a gross simplification. But how do you make progress unless you start with a simple alphabet and build from that, rather than start by being overwhelmed by the complexity?
AC: What you have brought up here I think is extremely interesting. Brodie was, of course, a very, very bright person, but he was really not at all burdened by too much knowledge about the brain. And he was so courageous to go right into it and to make this fantastic discovery that we have profited from so much; that was in a way, career. . . .
LS: Career openers?
AC: Career openers. That was very generous, in many ways, I must say. Not to digress, he was a very generous person.
LS: Yes, I think so. Just to return to this reward subject for a second (and to get just slightly historical and a little bit personal), when I left the Walter Reed I went to the VA Research Laboratories in Pittsburgh that were directed by a pharmacologist by the name of Amadeo Marrazzi. Another simplifier, Marrazzi had a simple electrophysiological transcallosal system and liked to analyze it, “Brodie-like,” in terms of the interplay of a couple of important transmitters. And I developed with the secretary of the ACNP, Oakley Ray, who was my first post-doctoral student, a sensitive self-stimulation technique where an animal traced out continuously its threshold for brain reinforcement. The rat operated two levers; one lever gave brain stimulations but the current came down in small steps with successive responses. When the current no longer was reinforcing, the animal would hit a second lever to reset the current back up to the original top reinforcing level. When plotted, the jagged edge of the reset curve (reflecting the sequence of reset currents) traced the approximate reward threshold. And, lo and behold, moderate doses of amphetamines lowered the threshold of reward and chlorpromazine, the opposite partner, elevated the threshold for reward. Other CNS drugs, such as the anticholinergic agent scopolamine or the anesthetic pentobarbital, had negligible effects on reward thresholds. Because amphetamine was known to release catecholamines and chlorpromazine to block catecholamine receptors, these results opened up my catecholamine theory of brain stimulation reinforcement. And if one assumes that brain self-stimulation is not just an artifact, but, in fact, that the map of reward sites reveals the brain substrate for natural reinforcement, then these findings support a catecholamine theory of both natural and drug-induced reinforcement. This theory has been a theme in my research for a long period of time. And, although I have closely followed your work, I probably could have been more attentive and saved myself some time if I had featured your important advances in dopamine pharmacology at an earlier point than I am doing now.
AC: Tell me a little bit about how you got into this area of catecholamines and a little bit more in detail what you have in mind when you mention dopamine here? Also, how you felt about the catecholamines when you started to focus on them?
LS: This is very much the Swedish connection. At the time that Oakley Ray and I were doing our studies with amphetamines and self-stimulation and trying to analyze the biochemical action of amphetamine, amphetamine’s action was not connected so robustly to dopamine as it is now. And, indeed, there is a Nobel Prize winner, who will remain unnamed, who felt that amphetamine’s behavioral actions were mediated in fact by serotonin. But this was in the early 1960s and our best brain stimulation electrodes were located laterally in the hypothalamus in a structure called the medial forebrain bundle. Animals would work very hard to stimulate their lateral hypothalamus and we also knew that they would work even harder for small currents if they had a little amphetamine. And then, lo and behold, in 1964, papers by Fuxe, Hökfelt and others appeared out of the Karolinska Institute showing these wonderful dopamine and norepinephrine fiber bundles running through the lateral hypothalamus on their way upstream to the limbic system and the neocortex. These fiber bundles were the obvious places where reward electrodes might be stimulating and a light went on. It just seemed very possible that those electrical stimulations that were so highly rewarding for the rat were activating catecholamine fibers and splashing catecholamines onto their cellular targets in the brain. So, that was the catecholamine theory of reward, formulated in the early days before there were good pharmacological tools for differentiating noradrenaline effects from dopamine effects. So, as a young man, I sadly emphasized only noradrenaline and could have taken a cue from you, Arvid, and given dopamine its rightful place.
AC: Well, I must say in this context that that you were not the only one to put emphasis on norepinephrine to start with; as you remember, dopamine has been, how is it called in English?
LS: The precursor?
AC: No, no, well, it will come a little later. At any event, it took a long time for dopamine to come into glory. I mean, it was maybe 10 years after the discovery of dopamine before people started to think that maybe there is something important about dopamine. So, what you had in mind was really quite in line with what people felt at that time.
LS: I had the advantage of doing an experiment in the early 1970s. I had moved from the VA to the drug industry, into a basic research job at Wyeth Laboratories. Curiously, Wyeth Laboratories did not make an explicit decision to hire a basic research psychopharmacologist to work in pure science. But this is what I wanted to do, and they were willing to support this activity, and indeed, they did so generously and I never had to worry about the funding of research for the many years I was there.
I had several collaborators at Wyeth, one of whom was Bruce Baxter, who did self-administration. In this type of experiment, the reinforcement is an injection of a drug (typically an intravenous injection) in place of the electrical brain stimulus. I realized that perhaps one way to settle the nasty little dopamine versus norepinephrine issue in the catecholamine reward theory was to offer a rat the dopamine receptor stimulant, apomorphine. You see, I knew that apomorphine treatments in dogs and in people can make them very sick and they throw up. Of course, the rats should not like apomorphine and should not self-administer this powerful dopamine receptor stimulant. So, one possible way to dispose of dopamine as a reward transmitter candidate was to test apomorphine in the self-administration experiment. I was so sure of the probable negative outcome of this experiment that, rather than troubling to set up the self-administration procedure in my own laboratory, we went downstairs into Bruce Baxter’s lab.
By God, I can still remember vividly Bruce Baxter coming up to me one day very excited with the tracings, showing me that the rats loved to self-administer apomorphine! That was a very big, early hint that dopamine should get more prominence in the reward theory. And if we have a chance to discuss my more recent cellular work, dopamine comes to full glory in these cellular reinforcement experiments.
AC: So, let’s do that for sure. Now, the name that I couldn’t remember a little while ago, of course, was “Cinderella.” Dopamine is the Cinderella of neuropharmacology.
LS: But we know how Cinderella ended up.
LS: She married the Prince.
AC: Yes. One thing that I think is kind of common in our work, yours and mine, is that we have focused on catecholamines, but then all of a sudden serotonin may show up. Tell me a little bit about how serotonin came into your work?
LS: Okay, again, I think, perhaps, we can flash back very briefly to that little diagram of Bernard Brodie and remember that the neuron that he drew (to represent the brain) had a serotonin input. I forget whether it was a plus sign or a minus sign, but I believe a minus sign, and it also had a norepinephrine input with the opposite sign. There was algebraic summation between these influences because these were antagonistic transmitters. And, the algebraic sum, or however the algebra went, determined the neuron’s behavior. So, this was the very simple theme when I started to consider my own theory of behavior. I needed to analyze the opposite paradigm to the reward paradigm, the so-called punishment paradigm, and the experimental work started with my collaborators at Wyeth Laboratories, Irving Geller and Joseph Seifter. These investigators devised a “conflict” procedure to measure punishment effects and to explore the pharmacology of the punishment system. This research was of practical interest to Wyeth because the conflict test was an excellent psychopharmacology screening method for detecting the anti-anxiety actions of meprobamate and benzodiazepines. Indeed, it turned out to be a very powerful predictive tool. But there was not a lot of understanding yet of the underlying biochemistry of either meprobamate or benzodiazepines. Then one day a paper appeared; Irv Kopin, I believe, was the senior author. My group, and other groups in Sweden, I think, then did related studies, all showing that the turnover of serotonin was markedly diminished by benzodiazepines. “Ah hah,” I said, looking at the Kopin paper, “Isn’t that interesting? If serotonin is the opposite number of catecholamines, which facilitate behavior through reward processes, then perhaps serotonin acts as the chemical brake which inhibits behavior through punishment processes. By reducing serotonin turnover, benzodiazepines release the brake and disinhibit the conflict behavior.”
We evaluated this idea by testing a large number of different serotonergic agents in the conflict procedure. The results satisfied me that, in fact, an important component of this admittedly very complicated serotonin system is to mediate behaviorally-inhibitory effects through a punishment mechanism. But with this background, it was quite surprising to me, Arvid, to see you pioneering the development of a novel antidepressant drug that increased serotonin activity by blockade of its reuptake. My simple little theory, based on the Brodie model, would have said, “Well, this drug of yours should rather have been a nice anxiety generating compound, hardly an antidepressant!” And so, it’s a good lesson again to remember that the brain certainly possesses the necessary complexity to account for the complexity of behavior. We must, perhaps, analyze one factor at a time and then try to study the interactions between different factors, but we should always be prepared for surprises. And so, the question of serotonin’s role in the action of antidepressants is, I think, a wonderful problem.
AC: But you were nearly right. I begin to sense that you found that serotonin and anxiety, the control of anxiety, are close to each other, somehow. Then you sort of turned it upside down, but still, you were pretty close, weren’t you?
LS: Well, I think that there is a component of the serotonin system that is involved in punishment. Punishment, however, may be distinguished from anxiety and so I am not sure if there aren’t also some serotonin systems that might increase anxiety. Afterthought: interestingly the chief adverse effects of Prozac and other SSRIs include anxiety and nervousness. But I definitely think that serotonin acts generally as an inhibitor of behavior. There is serotonergic inhibition of sexual behavior and of aggression. There is serotonergic inhibition of feeding; thus, many good anorexics are serotonergic. So, there seems to be a theme that behavioral inhibition, not necessarily brain inhibition, but behavioral inhibition, may have a common serotonergic basis. And it would be a wonderful synthesis and resolution if there were some way to connect behavioral inhibition to antidepressant action.
AC: Yes. Larry, you have been in the area of mood control and you have been in the area of anxiety, so tell me a little bit about schizophrenia now.
LS: Oh yes, schizophrenia. Schizophrenia, of course, is the elephant that all the blind men put their hands on. To some, schizophrenia is like a snake when you grab the tail; to others, it’s a wall when they press against the side. Schizophrenia is a fascinating problem and interesting experiment of nature in producing a distortion of some of the most important higher functions of the brain. Unfortunately, I don’t think investigators have yet put their finger on, in a really precise way, what the fundamental deficit in schizophrenia is. I think we need a more insightful clinical and behavioral analysis of schizophrenia in order to move the pharmacological and biochemical analysis. Perhaps we should talk about some of the ideas we have already discussed in terms of potential fundamental deficits in schizophrenia.
The schizophrenic symptom that was particularly attractive to me, as one of the blind men with a career-long interest in reward, was anhedonia. This symptom, of course, is one of Bleuler’s fundamental symptoms and, to me, the anhedonia immediately says impaired reward function. And so, shortly after the powerful catecholamine toxin, 6-hydroxydopamine was discovered, I got what I thought was a wonderful idea: Schizophrenia might be a neurodegenerative disease caused by an endogenous toxin, since it’s a long-term disorder with a chronic downhill course in many cases. Again, the anhedonia was what I focused on as well as a lack of purposefulness in the behavior, in other words, I focused on that behavior and thinking as not goal directed. All this implied to me a degenerative disorder of the brain reinforcement system. Clinical observations of euphoria in early schizophrenia additionally suggested a paradoxical initial disease phase involving a toxic stimulation of catecholamine reward functions. Such catecholamine hyperactivity could explain the acute antipsychotic effects of phenothiazine drugs, which, of course, are dopamine blockers. Put another way, early psychosis may represent a reward system out of control. But I was mainly interested in the chronic, deteriorating, longer-term phase of schizophrenia and conceived the hypothesis that through some enzymatic error in the schizophrenic brain, certain dopamine, and particularly norepinephrine, neurons might secrete 6-hydroxydopamine rather than their natural transmitter.
6-hydroxydopamine is a very toxic material. Perhaps acting as a false transmitter or metabolic product, 6-hydroxydopamine would be taken back up into the catecholamine nerve ending and would destroy it. I had the audacity to write a little theoretical paper, which incidentally was considered only for two weeks in the editorial offices of Science before acceptance. The paper included some interesting experimental results: if we gave rats 6-hydroxydopamine intraventricularly, we produced a catatonia as well as big deficits in self-stimulation and other reward behaviors. And chlorpromazine, which was known to protect against the neuronal uptake of 6-hydroxydopamine, prevented these deficits. So there were several pieces of data here to propose a 6-hydroxydopamine theory of schizophrenia.
This publication gave me my 15 minutes of fame. I was invited to speak everywhere, Arvid, but to my surprise, I quickly found out I was often invited to be criticized and to be shown wrong. I was still a relatively young man and I believe what I didn’t quite realize was that a lot of people looked at schizophrenia as a lifetime research occupation. They apparently were not ready for a solution at that time. In the early ‘70s a solution to the schizophrenia problem might perhaps put an untimely end to many grants and research careers. And so, I traveled to many places and took heavy criticism for this audacious speculation. We also did some post-mortem work and discovered – consistent with the theory – some enzyme deficits in the brains of schizophrenics. These findings were not replicated, however, and I think there may be a lesson here for some of the work that we see in the meeting today. With the new brain imaging procedures, etc., whenever one has a new machine, a new tool, or a new method, inevitably one makes some comparative measurements on schizophrenics. Of course, by chance, some differences will appear and these will get published. Then, they have to be chased down later.
AC: Is there a possibility that through oxidation there could be formed some toxic metabolites of catecholamines? This is still an area, of course, that is quite active, even though, perhaps, the 6-hydroxydopamine pathway isn’t so much looked upon now, but you have the oxidation pathway to form the very toxic quinones. And actually, we have in post-mortem brains of schizophrenics seen an elevation of these substances, so yes, you may have been right. Just like Brodie was right; although not exactly the way or not precisely in the sense that you formulate.
LS: Yes, and I think the other thing that is interesting along these lines is that the formation of neuromelanin may provide a protective pathway to dispose of these dangerous quinones. Incidentally, the names of the important catecholamine cell groups, the locus coeruleus (blue body) and the substantia nigra (black substance) are due to the neuromelanin content of these aggregates of dopamine and norepinephrine cell bodies.
AC: So, Larry, what do you think? Should we now move into what you are working on now, this fascinating work on what single cells can be doing in terms of reward and all that?
LS: Yes, that may be worth discussing, and this will take us into my current research. I begin by recognizing an important difference between voluntary and involuntary behavior. Only voluntary behavior can be controlled by rewards. This is because involuntary or reflex behavior is under the powerful and complete control of input stimulation; therefore, reflexes cannot be affected by rewarding outcomes or consequences. Reward processes permit successful behaviors to be built up and strengthened (reinforcement) and unsuccessful behaviors to be weakened or extinguished (non-reinforcement). Thus, these processes may have been decisive in the evolution of intelligent behavior.
Next, there is the very awkward question of the substrate of voluntary behavior. Most people think that complicated circuitries underlie all voluntary behavior; they assume, when the rat presses the lever that a lever-press circuit has fired off. But no one has yet found such a circuit, and I am not sure, if I were a young PhD student, that I would want to do a thesis where I had to identify such a circuit. In my opinion, current conceptions of how reward works are most unsatisfactory. These conceptions tend to emphasize the hedonic qualities of reward and they assume, in effect, that the “good feelings” generated by rewards can somehow reorganize and strengthen response circuitries. So, what we have here is a very casual, almost lay, type of notion.
And without tangible changes in behavioral circuitry to measure, how would one begin to assess the hedonic effects of reinforcement? After thinking about this difficult question for a long time, I found it useful to consider an alternative formulation. Incidentally, behavior is only arbitrarily broken into discrete responses; rather, like our stream of consciousness, behavior seems to flow continuously in constant interaction with the environment. If so, we may need a different conception of the behavioral substrate, perhaps some brain structure or more widespread collection of neurons that is spontaneously active. Such spontaneous neuronal activity could produce a continuous stream of behavior. If this idea were correct, then rewards might really act, not to rearrange brain circuitry, but instead to change the firing patterns of individual brain cells.
There is a way of testing such an idea. One could determine whether or not the activity of an individual neuron can be affected by reinforcement. Can one reward a neuron for, perhaps, firing off in a bursting pattern? The immediate difficulty is: how would one reward a brain cell? Our solution was based on the fact that cocaine is one of the most powerful pharmacological rewards known; the behavior of all animals tested, including humans, is reinforced by injections of this drug. Our cellular reinforcement experiments were performed in hippocampal slices to isolate the test neurons from the rest of the brain. We puffed minute injections of cocaine through the recording micropipette directly to the hippocampal cell whenever it performed the “correct” response, firing off in a bursting pattern. To our surprise, rates of bursting were progressively increased, not only by cocaine, but also by our good friend, the reinforcement transmitter dopamine. Dopamine was effective in a certain part of the hippocampus (CA1), whereas opioids (such as dynorphin) seemed to be effective in a different part of the hippocampus (CA3). Also, it was very significant that, just as in the case of whole-animal behavior, the burst-increasing effects of dopamine and dynorphin were observed to be “activity-dependent.” Thus, the drugs increased rates of bursting only if given when the target cell had recently been actively bursting. If the cell had been firing in single spikes or was silent, then the microinjections were ineffective or even tended to suppress bursting rates.
AC: So, now you have some very intriguing evidence that a single cell can have a reinforcing mechanism. May I ask you a somewhat philosophical question? Do you think a single cell could have a mind?
LS: A mind? My own personal, perhaps unsophisticated, view on the mind-body question perhaps, being alluded to here is: how can objective things like the brain and its cellular activity be translated into subjective things, such as the mind. My own treatment of the brain-mind problem is to say that it’s really a structure-function distinction. The mind should be thought of as a function of the brain, much as blood pressure is a function of the cardiovascular system. If this analogy is correct, then the fact that structure, brain, has functions, mind, is not a great puzzle and does not seem to constitute a philosophical dilemma. But I am not sure that everyone would be satisfied that subjective experience can be treated merely as a brain function and thus solve the problem. However, if one accepts my definition, and if one also assumes that a neuron can exhibit a reinforcement function, then one might wish to assign a subjective correlate to that cellular function. If so – and I say this with some difficulty – then maybe a cell does have a mind! I see what you have tapped into here.
But all this makes me a little bit uncomfortable because I was brought up in a very behavioristic tradition; to me mind is almost a naughty word. Perhaps we should not be so rigid in our behaviorism and it may be time to consider seriously such concepts as mind and consciousness. Perhaps someday we can do the correct brain imaging and image the mind.
AC: That brings me to my last question. The area that you have pioneered has been considerably important for drug development, so from your perspective can you tell me a little bit about what do you think is going to happen, in terms of drug development, in the next, let’s say, the next five years or so?
LS: Arvid, you do know how to ask difficult questions! I thought the question that was coming was: in this age of binding technology and molecular biology, what is the place of the older behavioral technologies which played such an important role in the development of many of our current drugs? Are we losing an important art with the somewhat diminishing interest in behavioral technologies? I do have a concern about that. Science follows fashions. Perhaps both of us can agree that the advances in molecular biology are wonderful and will help us to identify many of the receptors that we are interested in as academic pharmacologists. And in terms of practical drug development, molecular biology will produce some very interesting and wonderful natural protein hormones that might be advantageous. But molecular biology has to work with pharmacology to produce smaller molecules that might be useful as drugs which can be easily administered.
Maybe I shouldn’t back off the question of new developments, but it’s hard to confine oneself to a five-year time period because it takes so long to bring out a new drug. We still have a problem in the antidepressant field, where the drugs act too slowly, but fortunately, work such as yours with zimelidine has brought us a new class of antidepressant drugs that are largely free of many bothersome side effects. These side effects inhibited the use of antidepressants particularly in mild depressions because these patients are so fussy anyway; and if you poison them with anticholinergic and antihistamine effects and so forth, then they don’t stay on their medications. And so, the selective serotonin reuptake inhibitors, I think, are producing a revolution in the treatment of mild depression.
After many years of looking for ever more specific dopamine blocking agents, the recent recognition that a relatively nonspecific agent, clozapine, may be more effective than other anti-schizophrenic drugs is interesting. The search for new clozapine-like agents should advance the treatment of schizophrenia. Also, there is a lot of interest now in the biology of memory. An increase in the elderly population makes the treatment of Alzheimer’s disease, senility and other important ailments of memory more pressing; indeed, in depression, memory deficits are a common and most troublesome symptom. Hopefully, we will soon see advances in memory enhancement by new drugs.
One of the most exciting new discoveries may move us a little bit out of psychopharmacology proper into a more generally metabolic area. I am very excited about the new protein, leptin, which controls feeding behavior and induces satiety. Leptin evidently is a protein signal from the fat deposits that tells you how much fat you have and how heavy you are. This signal is compared with the setting in the brain which tells you what your ideal weight is. If you have too much fat, you will release a lot of leptin; then, you will eat less. But if you are not quite fat enough and release too little leptin, then you will eat more. There also are effects of leptin on fat metabolism, temperature, blood sugar regulation and so forth. So, in the area of feeding and weight control, a new hormone has been discovered with a target, very importantly, in the CNS. How it affects some of the other eating disorders, such as anorexia nervosa, may turn out to be very interesting.
AC: Well, thank you very much, Larry.
LS: Thank you so much, Professor Carlsson.
Larry Stein’s contributions to behavioral pharmacology and the development of neuroscience might be summarized under the six headings:
1. Positive reinforcement
2. Central mechanism of amphetamine
3. Which catecholamine is most reinforcing?
5. Opioid peptides
6. A cellular analog of operant conditioning
Stein’s contributions on the neurochemistry of positive reinforcement followed the discovery of brain self-stimulation by Olds and Milner in 1954, who had shown that the catecholamine antagonist, chlorpromazine, selectively suppressed self-stimulation behavior at doses which do not depress spontaneous motor activity (Olds and Milner 1954). But – paradoxically – Olds found, at the same time, that the catecholamine facilitator, amphetamine, also suppressed self-stimulation. By use of lower and more appropriate doses of amphetamine, Stein discovered that this catecholamine-enhancing drug will reliably, and often strikingly, increase the rate of self-stimulation (Stein 1964; Stein and Ray 1960). Furthermore, he was able to demonstrate for the first time that amphetamine facilitates self-stimulation behavior primarily by lowering the threshold current intensity for brain-stimulation reinforcement (Stein and Ray 1960). Just at this point in the early 1960s, Hillärp’s group at the Karolinska Institute in Sweden reported that most dopamine and norepinephrine fibers ascend in the brain in the lateral hypothalamic medial forebrain bundle. Since medial forebrain bundle electrodes had long been known from Olds’ work to support the very highest rates of self-stimulation, Stein put these anatomical facts together with his rudimentary but critical pharmacological observations, i.e., amphetamine-induced facilitation and chlorpromazine-induced suppression of self-stimulation. Taken together, these facts led Stein to propose a novel hypothesis that behavioral reinforcement is specialized neurochemically and is mediated by brain catecholamine systems (Stein 1962, 1968). Stein’s original catecholamine hypothesis of positive reinforcement has guided research in this field for more than 50 years. It has led fruitfully to more precise hypotheses that dopamine, norepinephrine, opioid peptide and cannabinoid systems may serve as important brain substrates for the reinforcement of operant behavior in animals and man. These ideas are now widely accepted and they are supported by a vast number of brain self-stimulation, drug self-administration, electrophysiological and optogenetic experiments from many laboratories all over the world (Wise 2008).
Central Mechanism of Amphetamine
Intrigued by the idea that the brain’s reinforcement system might be selectively facilitated by amphetamine, Stein next investigated the pharmacological mechanisms underlying amphetamine’s behavioral actions (Stein 1964). When these studies began, two opposing hypotheses of amphetamine’s central actions were under consideration: (#1) direct activation of brain catecholamine receptors and (#2) indirect activation of brain catecholamine receptors by drug-induced release of catecholamines. Many early studies had already indicated that amphetamine’s behavioral effects -- unlike its peripheral effects-- are unaffected (or even are potentiated by reserpine (Smith 1963). These initially unexpected observations supported the receptor activation hypothesis (#1) because reserpine depletes brain catecholamines and leaves little endogenous transmitter in neuronal stores for amphetamine to release. Thus, for amphetamine’s behavioral actions, the release hypothesis (#2) had little currency in 1964. Peter Dews had invited Stein to discuss his self-stimulation research in a high-visibility FASEB Symposium (Smith 1963). Stein’s original idea was to present some initial self-stimulation results which again suggested that amphetamine’s effects are undiminished after reserpine, indeed, he intended to support the receptor activation hypothesis (#1). But, in further experiments made shortly before the Dews’ symposium, Stein prudently assessed the effects of very large reserpine doses to ensure the near-total depletion of catecholamine stores. To his surprise, amphetamine’s effect on self-stimulation was now radically diminished in every rat tested (Smith 1963). (At present, it is known that amphetamine’s behavioral effects are generally unimpaired even after severe, but incomplete, depletion of catecholamines). The quantitative precision of the operant methodology encouraged Stein to conclude from his unanticipated observations in 1964 that amphetamine must exert its behavioral actions indirectly via the release of catecholamines. These results and conclusions have since been amply confirmed (Costa and Garattini 1970).
Which Catecholamine is Most Reinforcing?
For Stein, the question of which catecholamine is released by amphetamine (or positive brain stimulation) to produce behavioral reinforcement remained open in 1964: Dopamine and norepinephrine were the most likely candidates, but epinephrine could not be definitely excluded. Arguments favoring norepinephrine were: a) it is the substance released by amphetamine from sympathetic nerves in the periphery and b) it occurs in high concentrations throughout the reward system. Arguments favoring dopamine were: a) it is a much better substrate for monoamine oxidase than norepinephrine and b) it occurs in high concentrations in the extrapyramidal system, the final common pathway, perhaps, for the modulation of behavior by reinforcing stimuli (Stein 1964). Unfortunately, good pharmacological tools for the absolute discrimination of dopamine’s and norepinephrine’s behavioral actions were not available in this period. Self-stimulation data from Stein’s laboratory later revealed behavioral facilitation after intraventricular administrations of l-norepinephrine, but not of d-norepinephrine or dopamine (Wise, Berger and Stein 1973). Such findings led Stein initially to favor norepinephrine over dopamine as the most salient mediator of amphetamine-induced and self-stimulation reinforcement, but strong evidence favoring dopamine was rapidly accumulating (Wise 1982). In 1974 Stein realized that a stringent test of the dopamine hypothesis might be achieved by offering apomorphine (a powerful D2-dopamine receptor agonist) as reinforcement in drug self-administration tests. Small doses of apomorphine induce nausea and vomiting in many animal species, including man thus, if despite its potential adverse actions, apomorphine could be shown to reinforce self-administration behavior, the dopamine hypothesis would be strongly supported. In collaboration with Bruce Baxter and others at Wyeth responsible for the company’s ongoing self-administration screening activity, Stein found for the first time that rats will avidly self-administer apomorphine (Baxter, Gluckman, Stein and Scerni 1974). In 1992 Stein and his student David Self further demonstrated that rats will self-administer the D1 dopamine receptor agonist SKF 38,393 as reliably as they will self-administer D2-dopamine agonists (Self and Stein 1992). This observation refuted the view, widely held at the time, that D1-dopamine receptors are not involved in positive reinforcement.
Stein’s important studies of punishment system neurochemistry and the mechanism of action of benzodiazepines were initiated in 1970. Graeff and Schoenfeld had reported in 1970 very large increases in the punished response rates of pigeons after injections of serotonin antagonists (Graeff and Schoenfeld 1970). At about the same time, biochemical studies in many laboratories were revealing that benzodiazepines and related agents (barbiturates, meprobamate) substantially reduce the turnover of serotonin and norepinephrine in the brain. Previously, in 1968, Margules and Stein had found that the punishment-lessening and response-depressant effects of oxazepam followed opposite courses in the Geller-Seifter “conflict” test (Margules and Stein 1968). In confirmation of clinical observations, oxazepam’s depressant action (measured by a decrease of non-punished behavior) was observed to undergo tolerance after 3 to 6 doses, while its “antianxiety” action (measured by an increase in the rates of punished behavior) failed to show tolerance and often even increased after repeated drug administrations.
In 1972 Stein’s group integrated these behavioral and biochemical observations and reported that oxazepam’s effects on norepinephrine turnover rapidly undergoes tolerance, whereas its effects on serotonin turnover remains stable (Wise, Berger and Stein 1973). This led Stein and his co-workers to hypothesize that benzodiazepines may exert their punishment-lessening effects by a reduction in central serotonin activity (and their response-depressant effects by a reduction in central norepinephrine activity). Stein’s group also provided the first behavioral evidence (i.e., selective picrotoxin reversal of the punishment-lessening effects of benzodiazepines) in support of the now well-established view that benzodiazepines may influence monoamine turnover by secondary effects, which arise from a primary action on the gamma amino butyric acid (GABA)A receptor complex (Stein, Wise and Belluzzi 1977 – NO REF). Picrotoxin is a non-competitive antagonist for GABAA chloride channels. The serotonin-GABAA hypothesis of benzodiazepine’s behavioral actions has since been amply confirmed (Sepinwall and Cook 1980).
The first identification in brain of natural opiate receptor agonists — met-enkephalin and leu-enkephalin — impacted pharmacology and neuroscience in 1975 with a force similar-to that exerted on physics by the discovery of radioactivity at the end of the 19th century. For months, neuropharmacologists were engrossed with little else and Stein was no exception. Since enkephalins are pentapeptides, with poor brain penetration, it was necessary to inject them directly into the brain to study their behavioral actions. In 1976-77 Stein’s group reported two studies in Nature which demonstrated for the first time that: 1) “methionine-enkephalin and leucine-enkephalin, when administered through permanently-indwelling cannulae in the lateral ventricles of rats, induce a profound analgesia in vivo that is fully reversible by naloxone” and 2) “that rats will work for enkephalin injections delivered directly into the ventricles of their own brains” (Belluzzi, Grant, Garsky et al. 1976; Belluzzi and Stein 1977). Of particular-interest, in the light of the generally superior potency of the methionine peptide, is the observation that leucine-enkephalin is self-administered with greater avidity than methionine-enkephalin” (Belluzzi and Stein 1977). Perhaps unsurprisingly, these papers on enkephalin — as well as an anatomical mapping study on the distribution of enkephalin-immunoreactive cell bodies in the rat brain performed with Tomas Hökfelt and others — are among the most frequently cited articles in Stein’s bibliography (Hökfelt, Elder, Johansson et al. 1977).
A Cellular Analog of Operant Conditioning
Stein’s latest work concerns the most difficult question that he addressed over his long career: Which brain structures are the most likely to receive signals from (and thus be reinforced by) the dopamine and opioid-peptide reinforcement systems. Since it is chiefly operant behavior that is reinforced, it is plausible to assume that behavior-controlling brain substrates are major targets of the reinforcing systems. But little or nothing is known about the actual neural substrates of operant behavior.
It is commonly believed that positive reinforcement is exerted at the systems level and probably involves the strengthening or reorganization of complex “whole-response” circuitries. But whether or not particular behavioral variations are treated as the same or as different responses depends on the reinforcement contingencies. Thus, for example, if lever-press responses of 5-g and 10-g force are reinforced indiscriminately, both are counted as the same “correct” response; however, if they are selectively reinforced, the behavioral variations clearly are regarded as different responses. The fact that closely similar behavioral variations may be reinforced either indiscriminately or selectively is presumptive evidence that the unit of reinforcement cannot be the whole response itself. B.F. Skinner has deeply considered these problems and has concluded that the operant response must be a composite made up of elements and that it is these “response elements” or “behavioral atoms” which are the more likely functional units for reinforcement (Skinner 1953).
In a first and very preliminary approach, Stein has assumed that Skinner’s behavioral atoms might conceivably be represented at the neuronal level by individual cellular responses. Using a brain slice preparation, he has attempted to demonstrate -- in the absence of most of the brain -- the in vitro operant conditioning of hippocampal bursting activity using micropressure applications of dopamine, cocaine or other behaviorally-reinforcing agents directly to the cell body as cellular reinforcements. The in vitro reinforcement test employs training procedures closely analogous to those of behavioral operant conditioning. The most important, and indeed defining, feature of behavioral operant conditioning is an absolute requirement for response-reinforcement contingency. Accordingly, in Stein’s cellular analog, it was obligatory to show that only burst-contingent (and not burst-independent) micro-applications of reinforcing drugs will produce significant enhancement of hippocampal bursting.
Stein and his collaborators observed that the bursting responses of individual CA1 pyramidal neurons were progressively increased in a dose-related manner by burst-contingent micro-puffs of dopamine and cocaine, whereas the bursting responses of CA3 units similarly were increased by morphine and dynorphin (Stein, Xue and Belluzzi 1973, 1974). The same microinjections, administered independently of cellular bursting, failed to facilitate and frequently suppressed CA1 and CA3 bursting, respectively — this observation suggested that nonspecific stimulation of cellular activity is an unlikely explanation of the facilitatory action of the burst-contingent injections. Experiments with glutamate, an excitatory transmitter that is not commonly associated with the behaviorally-reinforcing effects of drugs, also contradicted the nonspecific-stimulation hypothesis. Burst-contingent injections of glutamate always failed to increase CA1 bursting.
All of these observations were consistent with the possibility that the bursting of hippocampal cells may be “operantly conditioned in vitro” by burst-contingent microinjections of behaviorally-reinforcing transmitters or drugs. Hence, on the basis of these observations, Stein, Xue and Belluzzi concluded: “If so, and if we have actually managed to put ‘a single neuron in a Skinner box’ …we may have had the good luck to get a glimpse of a Skinnerian behavioral atom” (Stein, Xue and Belluzzi 1974).
Over his long career of about 50 years Larry made several important experimental and theoretical contributions to behavioral pharmacology. Especially noteworthy are his pioneering investigations on the catecholamine mediation of operant reinforcement, serotonergic mediation of punishment, indirect (catecholamine-releasing) central actions of amphetamine and the first demonstration of the analgesic and reinforcing effects of enkephalins. His published work has had high impact on behavioral pharmacology — as one indication, among his peer-reviewed papers, are 17 Science and Nature articles. His catecholamine hypothesis of positive reinforcement has guided research on behavioral and stimulant drug reinforcement for 50 years.
Larry was an active contributing member of our International Network for the History of Neuropsychopharmacology (INHN). His Curriculum Vitae, Bibliography and Interactions in our exchanges will remain accessible on our website, INHN.org. He was the last surviving member of our pioneers in behavior pharmacology and his passing will leave a vacuum that cannot be filled. He will be missed by his family, friends and all of us who were close to him.
Ban TA. Preface. In: Shorter E, editor. Starting Up. Brentwood: American College of Neuropsychopharmacology; 2011a, pp. xxv -xl.
Ban TA, editor. An Oral History of Neuropsychopharmacology (10 volumes). Brentwood: American College of Neuropsychopharmacology; 2011b
Baxter B, Gluckman MI, Stein L, Scerni RA. Self-injection of apomorphine in the rat. Positive reinforcement by a dopamine receptor stimulant. Pharm Bioch Behav 1974;2:387-91.
Belluzzi JD, Grant N, Garsky V, Sarantakes D, Wise CD, Stein L. Analgesia induced in vivo by central administration of enkephalin in rat. Nature 1976;260:625-6.
Belluzzi JD, Stein L. Enkephalin may mediate euphoria and drive reduction reward. Nature 1977;266:556-8.
Brady JV. Assessment of drug effects on emotional behavior. Science 1956;123:1033 -4.
Costa E, Garattini S, editors. Amphetamines and Related Compounds. New York: Raven Press; 1970.
Graeff FG, Schoenfeld RI. Tryptaminergic mechanisms in punished and non-punished behavior. J Pharmacol Exp Therap 1970;173:277-83.
Hökfelt T, Elder, Johansson O, Terenius L, Stein L. The distribution of enkephalin-immunoreactive cell bodies in the rat central nervous system. Neuroscience Lett. 1977;5:25-31.
Margules DL, Stein L. Facilitation of Sidman avoidance behavior by positive brain stimulation. J Comp Physiol Psychol. 1968;66(1):182-4.
Olds J, Milner P. Positive reinforcement produced by electrical stimulation of septal region and other regions of the brain. J Comp Physiol Psychol. 1954;47(6):419-27.
Self DW, Stein L. The D1 agonists SKF 82958 and SKF 7754 are self-administered by rats. Brain Research 1992; 582:349-52.
Sepinwall J, Cook L. Mechanism of action of the benzodiazepines: behavioral aspect. Fed Proc.1980;39:3024-31.
Shorter E. Introduction and dramatis personae. In: Shorter E, editor. Starting Up. Brentwood: American College of Neuropsychopharmacology; 2011a
Shorter E, editor. Starting Up. Brentwood: American College of Neuropsychopharmacology; 2011b.
Skinner BF. Science and Human Behavior. New York: Free Press; 1953.
Smith CB. Enhancement by reserpine and alpha-methyl-dopa the effects of D-amphetamine upon the locomotor activity of mice. J Pharmacol Exp Ther. 1963;142:343-50.
Spence KW. Theoretical interpretations of learning. In Stevens SS, editor. Handbook of Experimental Psychology. Oxford: Wiley; 1951, pp. 690 -729.
Stein L. Effects and interactions of imipramine, chlorpromazine, reserpine and basis of depression. In: Wortis J, editor. Recent Advances in Biological Psychiatry. New York: Plenum; 1962, pp 288-308.
Stein L. Self-stimulation of the brain and the central stimulant action of amphetamine. Fed Proc 1964;23:836-50.
Stein L. Animal behavior and drug action. In: Steinberg H, deReuck AVS, Knight J, editors. London: J. & A. Churchill, Ltd. 1964, pp 91-118.
Stein L. Chemistry of reward and punishment. In: Efron, DH, editor. Proceedings of the American College of Neuropsychopharmacology. Washington: US Government Printing Office; 1968, pp. 105 -23.
Stein L. Interviewed by Arvid Carlsson. In: Shorter E, editor. Starting Up. Brentwood: American College of Neuropsychopharmacology; 2011, pp 177-88.
Stein L, Ray OS. Brain stimulation reward “Thresholds” self-determined in rat. Psychopharmacologia. 1960;1:251-6.
Stein L, Wise CD. Possible etiology of schizophrenia: Progressive damage to the noradrenergic reward system by 6-hydroxydopamine. Science 1971;171:1032–6.
Stein L, Wise CD, Belluzzi JD. Effects of benzodiazepines on central serotonergic mechanisms. In: Costa E, Greengard P, editors. Mechanism of Action of Benzodiazepines. Raven Press, New York; 1975.
Stein L, Xue BG, Belluzzi JD. A cellular analogue of operant conditioning J Exp Anal Behav 1993;60:41-53.
Stein L, Xue BG, Belluzzi JD. In vitro reinforcement of hippocampal bursting: a search for Skinner’s atoms of behavior. J Exp Anal Behav 1994;61:155-68.
Wise RA. Neuroleptics and operant behavior: the anhedonia hypothesis. Behav Brain Sci. 1982;5;39-53.
Wise RA. Dopamine and reward: the anhedonia hypothesis 30 years on. Neurotox Res. 2008;14: 169-83.
Wise CD, Berger BD, Stein L. Evidence of noradrenergic reward receptors and serotonergic punishment receptors in rat brain. Biol Psychiatry 1973;6:3-21.
October 17, 2019