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General Discussion
GENERAL DISCUSSION. President, Professor B. Scharrer REES:I would like to direct a question to Dr. Papaikonomou. How could you measure changing variables, for instance a change in the sensitivity of the pituitary gland to CRF? I cannot understand that with a change in the peripheral level of glucocorticoids the pituitary gland may be inhibited. Then you get at the same time a decrease in sensitivity to CRF. In this way you may have many variables.
VAN
PAPAIKONOMOU: You asked a question about experimental procedures, but I just made some computer calculations on the basis of literature data. When you have an isolated pituitary and you infuse CRF, then you obtain a particular curve which shows specific changes upon an increase and decrease of CRF. REES:May I make myself a bit clearer? When I listened to your paper I was asking myself how do you put in the many variables in your computer program?
VAN
I have not done this up to this time: what I plan to do is to employ PAPAIKONOMOU: the microcannulation technique developed by Porter by which you can infuse in the portal vessels at the same time CRF and corticosteroids. One can then measure the output of the hypophysis and at the same time the output of the adrenal gland. Then, by using the computer model of the adrenal cortex, you may obtain an idea of the interaction of the various systems. SLOPER: What is the question you are actually asking the computer? Why not use rats? PAPAIKONOMOU: I am using the computer in order to predict results by using data from the literature. In that way I may be able to design some specific experiments. DONOVAN: What Prof. Sloper is really asking is: if you get the data, why bother with the computer, since you have the physiological data anyway. Will these studies with a computer tell you what the physiological experiments are unable to do? SMELIK: In trying to study the hypophysis and adrenal from the viewpoint of biocybernetics you have the risk that you put more into the computer than comes out of it. 1 think the idea is that if you would be able to make an operational model, a simulation of the system, then you can predict several functions of the system which cannot be found experimentally. There are always aspects of the system which are very difficult or impossible to measure experimentally. One could imagine that it would be possible to get answers from the model which you cannot get in another way in the animal. From the computer results one can draw conclusions which can be
tested experimentally in the animal. I think it is good for all of us to look at the system in this way. DONOVAN: Perhaps the weakness of what you have been saying is that you made theoretical predictions with a computer. For these, in order to be valuable, have to be tested experimentally. If they cannot be tested experimentally, there are some hazards in relying on them. SMELIK:There have been, in the past decade, discussions about the setpoint of the adrenal system whether this would be a fixed setpoint or a variable one. What Dr. Papaikonomou tried to show was that just by a theoretical approach this setpoint is actually an operating stable point of the system and that this can be influenced by the conditions of the system. SCHARRER: It is a very complex situation which has been analysed here because we cannot really look at a system, such as the hypothalamo-hypophysial, without relating it with the adrenal and several other actions. I am wondering if, for those of us who are such beginners in trying to understand the value of such computer methods in biology, the speaker could tell us what the very simple or relatively simple system is, which has benefitted from such analysis. Or is this sort of contradictory in itself? PAPAIKONOMOU: The work of Starck in the USA who studied the pupil reflex mechanism.
You could not tell us just in similar terms how it is possible to relate SCHARRER: observations to computer reasoning or computer data?
I have used a computer program which simulates the adrenal cortex. PAPAIKONOMOU: I decided to make a model that can reproduce the dynamic characteristics of the adrenal cortex. The data for the model were real experimental data; of course, I had to make some postulations and programs in the computer and then look back to see whether the model was performing the same as results from the experimental studies. SCHARRER: That is part of the problem that faces the experimentalist, such as Dr. Donovan who feels that a theoretic consideration, unless it can be thoroughly tested and is parallelled by an experimental investigation, would give us a kind of theoretical biology which would be outside of our daily scientific armamentarium. SCHADE:The computer in biology has been used along two different lines. The first line of approach which is very well accepted, is the use of the computer as a calculation machine in order to process experimental data. A more difficult field is the application of systems analysis to the study of neuroendocrinology. The advantage of this method is that you can make predictions which you can use to reduce experimental data.
347 The application of the computer as a calculation machine is now very widespread.
SCHARRER:I wonder if somebody wants to bring up an entirely different or partly different subject. HAAR: I would like to bring up the question of axonal transport. You have a system here, whereby the cell can absorb chemicals and make these into larger molecules which cannot get out the way the substance came in. They have to be pushed down by whatever medium is available, and then they come out at the other end of the axon. TER
SCHARRER:In the transport idea, microtubules are involved in the transport of secretory granules. This holds not only for neural secretory but also for other secretory granules. The movement takes place by a spiral rotation on the outside of specialized structured microtubular surfaces. Part of the membrane of the granules is supposed to be engaged in such a way on the surface that it comes finally out on the end. So that would be a mechanism which needs some testing, but it has nothing, I think, to do with the size of the molecules. The molecules could get anywhere, but they want to get in the neuron to the end of the cell, because that is where the release side is. SMELIK:Many of us are simple endocrinologists, but we have been forced to think about the central nervous system because i t appeared that the endocrine system is controlled by the nervous system and so we get involved in all sorts of difficult things concerning neuronal and neurosecretory mechanisms. What we have tried to learn from the neurophysiologists and neuroanatomists is something about neuronal circuitry, synaptic transmission, action potentials, etc. Many of us remember the time that we thought of the hypothalamus system as a kind of supermaster gland for the endocrine system. If you want to know whether that gland produces any hormone, then, in the classical approach, you destroy that gland and/or make an extract from it and you inject it into an animal in which that structure has been destroyed. I think that, after a while, at least the endocrinologists who were going to call themselves neuroendocrinologists became aware of the fact that there are many networks in the nervous system. Now we believe that there are many systems which are involved in endocrine mechanisms and we think in terms of synaptic transmission. Many people in the past who have been working on the autonomic innervation of organs in the body have always advocated the idea that innervation of peripheral organs is not of a strict synaptic type, but that the products released by these nerves would act more as a sort of tissue hormone, influencing a whole area in a very diffuse way. In one of the discussions I have said something about the so-called adrenergic inhibition of ACTH control. What strikes me is that if you look at the adrenergic nerves in the brain, especially in the hypothalamus, then you see a network of very fine nerve fibres, everywhere. if you have a close look at one fibre then it looks like a chain of pearls and it is said that these could be all “en passant” synaptic contacts. If this is true you get the impression that every cell must be innervated in this way.
34s
My question comes to this; could it be possible that such a thing as has been found in the periphery could also be located within the central nervous system? SCHARRER: You made yourself clear, Dr. Smelik, and I can give you a very straight answer as far as I am concerned but I would like not to do that before I call on some of the more neurally oriented members of the audience. KAPPERS:The “pearls” you mentioned are the so-called varicosities of the axons, which contain an accumulation of the specific transmitter substance just as the axon terminals do. Their ultrastructure is also quite comparable to that of the axon endings. It is generally accepted that the neurotransmitter substance is released from these varicosities just as it is from the axon terminals. In the periphery, the varicosities of the sympathetic axons, as a rule, do not take part in the formation of classical synaptic structures. The transmitter substance, once depleted, diffuses in the tissue and exerts an influence on effector cells which may well lie at some distance of the varicosities. Axon terminals of sympathetic fibres in the periphery may or may not always form true synapses with effector cells. Very probably, the same will hold in the central nervous system. DROOGLEEVER FORTUYN: I have been taught that the brain was a reflex organism. When you have a certain input, you could expect a certain output. Now I am in the process of changing my basic concept. The brain apparently is not an instrument in which you can put in something and always get an output, since that depends on the internal state of the machinery. Hormones and other substances are now supposed to be able to change the sensitivity of the system and I heard that the sensitivity to estrogens is different on different times of the day and in different seasons of the year. Microelectrode work would not be possible if many cells would be influenced in the same way, because in general very specific response patterns are being found. So I don’t believe in the concept that some neurotransmittors or other substances may have a very diffuse effect upon the neurons in the central nervous system. SCEIARRER: I would like to make a very strong and clearcut statement about what we now call the neuron doctrine, which means that the nervous system consists of individual cells and not of a so-called terminal reticulum. The idea of the terminal reticulum is really abandoned. We now accept the fact that neurons interact by contact with each other and interact with other organs or parts of organs by letting a chemical mediator transfuse between the ending and the opposing membrane. HAYWARD: We must not be too impatient with the nervous system and its complexity. I would want to call your attention to the places that you can look for to get models but real models of the kinds of mechanisms that could be working in the hypothalamus. And Dr. SchadC has given you one group of model systems, namely Aplysia. In these animals one can study series of cells and their interactions and measure the membrane potentials which can give you some idea of how a group of cells would work. Perhaps
349 more pertinent to the hypothalamus is the research of the sympathetic ganglia in which the time course of excitatory and inhibitory potentials can occur in great distances of time. There are synaptic mechanisms that are available to explain many of the events that are occurring in the hypothalamus and it is just a matter of trying to get the experimental data for the particular mammalian system, as difficult as it will be to get, to try to document exactly what is happening. We arejust on the threshold ofbeginning to approach some of the basic questions of the hypothalamic mechanism. The basic neural mechanisms are being worked out and it is merely necessary to apply the techniques from Aplysia and from sympathetic ganglia to the hypothalamus. SMELIK:But even this long excitation or inhibition is a matter of seconds at most. If you give a single shot of progesterone and if you look 2 hours later you find somewhere in the reticular formation a lowering of the threshold value for electrical stimulation. Twenty-four hours later you find just the reverse, which means that there is an action of a hormone on certain neurons which is biphasic and lasting, at least about 24 hours. What sort of mechanism are you thinking of, then? Have you any idea what this could mean, where this influence takes place, because I don’t think that this has anything to do with synaptic mediators.
HAYWARD:I think Dr. Donovan has given us in his talk the kinds of sequences or events that can lead to various delays. If he is given a substance like estrogen, it is in fact prostaglandin which is setting up a sequence of events, which takes time. If you have given a hormone and you look at the total behaviour ofyour animal, progesterone is affecting every cell in the body, in the brain as well as in the rest of the body. It is like the slide I showed: brain stimulation produces many things and since we are looking for one system, we don’t pay attention to 4 or 5 others. So one may get a misleading picture. S C H A D ~There : is one set of data which may be of some relevance to this: if you inject a substance which directly affects the membrane pump, then the effect may only be observed after some 50 or 60 thousand action potentials. This may explain the various types of delay. DONOVAN:I have been thinking about the dopaminergic system within the hypothalamus. It struck me that this is being regarded as one system. Never have I heard any suggestion that there may be subsystems, maybe with the same chemical activities, each concerned with a different function controlling a different hormone. SMELIK:We are all convinced now that drugs or hormones might influence a great number of neurons at the same time and we accept the idea but it is very difficult to conceptualize how this is done and what really happens here. These are all things which have to be adapted to the concept of circuitry and synaptic transmission.
SCHARRER:I would like to come back to something that was discussed long ago.
350
It is a problem which faces the neuroendocrinologists, namely the question of the timecourse in the communication between neurons and nonneuronal elements, let us say endocrine effector cells. I hope to bring a little bit of life into that situation by saying that we have learned that the neuron by using specific chemical mediators which go far beyond those that act like the classic neurotransmitters, has learned to cope with these problems. In two ways, first by sending out hormone-like substances which have a different timecourse and by doing it in what I called a sort of intermediate way which does not need capillary systems or portal systems or the general circulation, that is in getting to adjacent cells in a sort of slow and diffused way. For this I brought into the picture those cases where we have stroma in between, or call it ground substance. Such things do exist in certain cases. Your question whether this occurs in the central nervous system is at the moment not to be answered in the affirmative because we have no basis for it. The trouble is that you are right in supposing something of a transmitter type action which lasts a thousandth of a second and does not serve our purposes well. But we have other methods, the neuron is more versatile than the classic neurophysiologists needed to deal with.
VAN
PAPAIKONOMOU: You asked a question about experimental procedures, but I just made some computer calculations on the basis of literature data. When you have an isolated pituitary and you infuse CRF, then you obtain a particular curve which shows specific changes upon an increase and decrease of CRF. REES:May I make myself a bit clearer? When I listened to your paper I was asking myself how do you put in the many variables in your computer program?
VAN
I have not done this up to this time: what I plan to do is to employ PAPAIKONOMOU: the microcannulation technique developed by Porter by which you can infuse in the portal vessels at the same time CRF and corticosteroids. One can then measure the output of the hypophysis and at the same time the output of the adrenal gland. Then, by using the computer model of the adrenal cortex, you may obtain an idea of the interaction of the various systems. SLOPER: What is the question you are actually asking the computer? Why not use rats? PAPAIKONOMOU: I am using the computer in order to predict results by using data from the literature. In that way I may be able to design some specific experiments. DONOVAN: What Prof. Sloper is really asking is: if you get the data, why bother with the computer, since you have the physiological data anyway. Will these studies with a computer tell you what the physiological experiments are unable to do? SMELIK: In trying to study the hypophysis and adrenal from the viewpoint of biocybernetics you have the risk that you put more into the computer than comes out of it. 1 think the idea is that if you would be able to make an operational model, a simulation of the system, then you can predict several functions of the system which cannot be found experimentally. There are always aspects of the system which are very difficult or impossible to measure experimentally. One could imagine that it would be possible to get answers from the model which you cannot get in another way in the animal. From the computer results one can draw conclusions which can be
tested experimentally in the animal. I think it is good for all of us to look at the system in this way. DONOVAN: Perhaps the weakness of what you have been saying is that you made theoretical predictions with a computer. For these, in order to be valuable, have to be tested experimentally. If they cannot be tested experimentally, there are some hazards in relying on them. SMELIK:There have been, in the past decade, discussions about the setpoint of the adrenal system whether this would be a fixed setpoint or a variable one. What Dr. Papaikonomou tried to show was that just by a theoretical approach this setpoint is actually an operating stable point of the system and that this can be influenced by the conditions of the system. SCHARRER: It is a very complex situation which has been analysed here because we cannot really look at a system, such as the hypothalamo-hypophysial, without relating it with the adrenal and several other actions. I am wondering if, for those of us who are such beginners in trying to understand the value of such computer methods in biology, the speaker could tell us what the very simple or relatively simple system is, which has benefitted from such analysis. Or is this sort of contradictory in itself? PAPAIKONOMOU: The work of Starck in the USA who studied the pupil reflex mechanism.
You could not tell us just in similar terms how it is possible to relate SCHARRER: observations to computer reasoning or computer data?
I have used a computer program which simulates the adrenal cortex. PAPAIKONOMOU: I decided to make a model that can reproduce the dynamic characteristics of the adrenal cortex. The data for the model were real experimental data; of course, I had to make some postulations and programs in the computer and then look back to see whether the model was performing the same as results from the experimental studies. SCHARRER: That is part of the problem that faces the experimentalist, such as Dr. Donovan who feels that a theoretic consideration, unless it can be thoroughly tested and is parallelled by an experimental investigation, would give us a kind of theoretical biology which would be outside of our daily scientific armamentarium. SCHADE:The computer in biology has been used along two different lines. The first line of approach which is very well accepted, is the use of the computer as a calculation machine in order to process experimental data. A more difficult field is the application of systems analysis to the study of neuroendocrinology. The advantage of this method is that you can make predictions which you can use to reduce experimental data.
347 The application of the computer as a calculation machine is now very widespread.
SCHARRER:I wonder if somebody wants to bring up an entirely different or partly different subject. HAAR: I would like to bring up the question of axonal transport. You have a system here, whereby the cell can absorb chemicals and make these into larger molecules which cannot get out the way the substance came in. They have to be pushed down by whatever medium is available, and then they come out at the other end of the axon. TER
SCHARRER:In the transport idea, microtubules are involved in the transport of secretory granules. This holds not only for neural secretory but also for other secretory granules. The movement takes place by a spiral rotation on the outside of specialized structured microtubular surfaces. Part of the membrane of the granules is supposed to be engaged in such a way on the surface that it comes finally out on the end. So that would be a mechanism which needs some testing, but it has nothing, I think, to do with the size of the molecules. The molecules could get anywhere, but they want to get in the neuron to the end of the cell, because that is where the release side is. SMELIK:Many of us are simple endocrinologists, but we have been forced to think about the central nervous system because i t appeared that the endocrine system is controlled by the nervous system and so we get involved in all sorts of difficult things concerning neuronal and neurosecretory mechanisms. What we have tried to learn from the neurophysiologists and neuroanatomists is something about neuronal circuitry, synaptic transmission, action potentials, etc. Many of us remember the time that we thought of the hypothalamus system as a kind of supermaster gland for the endocrine system. If you want to know whether that gland produces any hormone, then, in the classical approach, you destroy that gland and/or make an extract from it and you inject it into an animal in which that structure has been destroyed. I think that, after a while, at least the endocrinologists who were going to call themselves neuroendocrinologists became aware of the fact that there are many networks in the nervous system. Now we believe that there are many systems which are involved in endocrine mechanisms and we think in terms of synaptic transmission. Many people in the past who have been working on the autonomic innervation of organs in the body have always advocated the idea that innervation of peripheral organs is not of a strict synaptic type, but that the products released by these nerves would act more as a sort of tissue hormone, influencing a whole area in a very diffuse way. In one of the discussions I have said something about the so-called adrenergic inhibition of ACTH control. What strikes me is that if you look at the adrenergic nerves in the brain, especially in the hypothalamus, then you see a network of very fine nerve fibres, everywhere. if you have a close look at one fibre then it looks like a chain of pearls and it is said that these could be all “en passant” synaptic contacts. If this is true you get the impression that every cell must be innervated in this way.
34s
My question comes to this; could it be possible that such a thing as has been found in the periphery could also be located within the central nervous system? SCHARRER: You made yourself clear, Dr. Smelik, and I can give you a very straight answer as far as I am concerned but I would like not to do that before I call on some of the more neurally oriented members of the audience. KAPPERS:The “pearls” you mentioned are the so-called varicosities of the axons, which contain an accumulation of the specific transmitter substance just as the axon terminals do. Their ultrastructure is also quite comparable to that of the axon endings. It is generally accepted that the neurotransmitter substance is released from these varicosities just as it is from the axon terminals. In the periphery, the varicosities of the sympathetic axons, as a rule, do not take part in the formation of classical synaptic structures. The transmitter substance, once depleted, diffuses in the tissue and exerts an influence on effector cells which may well lie at some distance of the varicosities. Axon terminals of sympathetic fibres in the periphery may or may not always form true synapses with effector cells. Very probably, the same will hold in the central nervous system. DROOGLEEVER FORTUYN: I have been taught that the brain was a reflex organism. When you have a certain input, you could expect a certain output. Now I am in the process of changing my basic concept. The brain apparently is not an instrument in which you can put in something and always get an output, since that depends on the internal state of the machinery. Hormones and other substances are now supposed to be able to change the sensitivity of the system and I heard that the sensitivity to estrogens is different on different times of the day and in different seasons of the year. Microelectrode work would not be possible if many cells would be influenced in the same way, because in general very specific response patterns are being found. So I don’t believe in the concept that some neurotransmittors or other substances may have a very diffuse effect upon the neurons in the central nervous system. SCEIARRER: I would like to make a very strong and clearcut statement about what we now call the neuron doctrine, which means that the nervous system consists of individual cells and not of a so-called terminal reticulum. The idea of the terminal reticulum is really abandoned. We now accept the fact that neurons interact by contact with each other and interact with other organs or parts of organs by letting a chemical mediator transfuse between the ending and the opposing membrane. HAYWARD: We must not be too impatient with the nervous system and its complexity. I would want to call your attention to the places that you can look for to get models but real models of the kinds of mechanisms that could be working in the hypothalamus. And Dr. SchadC has given you one group of model systems, namely Aplysia. In these animals one can study series of cells and their interactions and measure the membrane potentials which can give you some idea of how a group of cells would work. Perhaps
349 more pertinent to the hypothalamus is the research of the sympathetic ganglia in which the time course of excitatory and inhibitory potentials can occur in great distances of time. There are synaptic mechanisms that are available to explain many of the events that are occurring in the hypothalamus and it is just a matter of trying to get the experimental data for the particular mammalian system, as difficult as it will be to get, to try to document exactly what is happening. We arejust on the threshold ofbeginning to approach some of the basic questions of the hypothalamic mechanism. The basic neural mechanisms are being worked out and it is merely necessary to apply the techniques from Aplysia and from sympathetic ganglia to the hypothalamus. SMELIK:But even this long excitation or inhibition is a matter of seconds at most. If you give a single shot of progesterone and if you look 2 hours later you find somewhere in the reticular formation a lowering of the threshold value for electrical stimulation. Twenty-four hours later you find just the reverse, which means that there is an action of a hormone on certain neurons which is biphasic and lasting, at least about 24 hours. What sort of mechanism are you thinking of, then? Have you any idea what this could mean, where this influence takes place, because I don’t think that this has anything to do with synaptic mediators.
HAYWARD:I think Dr. Donovan has given us in his talk the kinds of sequences or events that can lead to various delays. If he is given a substance like estrogen, it is in fact prostaglandin which is setting up a sequence of events, which takes time. If you have given a hormone and you look at the total behaviour ofyour animal, progesterone is affecting every cell in the body, in the brain as well as in the rest of the body. It is like the slide I showed: brain stimulation produces many things and since we are looking for one system, we don’t pay attention to 4 or 5 others. So one may get a misleading picture. S C H A D ~There : is one set of data which may be of some relevance to this: if you inject a substance which directly affects the membrane pump, then the effect may only be observed after some 50 or 60 thousand action potentials. This may explain the various types of delay. DONOVAN:I have been thinking about the dopaminergic system within the hypothalamus. It struck me that this is being regarded as one system. Never have I heard any suggestion that there may be subsystems, maybe with the same chemical activities, each concerned with a different function controlling a different hormone. SMELIK:We are all convinced now that drugs or hormones might influence a great number of neurons at the same time and we accept the idea but it is very difficult to conceptualize how this is done and what really happens here. These are all things which have to be adapted to the concept of circuitry and synaptic transmission.
SCHARRER:I would like to come back to something that was discussed long ago.
350
It is a problem which faces the neuroendocrinologists, namely the question of the timecourse in the communication between neurons and nonneuronal elements, let us say endocrine effector cells. I hope to bring a little bit of life into that situation by saying that we have learned that the neuron by using specific chemical mediators which go far beyond those that act like the classic neurotransmitters, has learned to cope with these problems. In two ways, first by sending out hormone-like substances which have a different timecourse and by doing it in what I called a sort of intermediate way which does not need capillary systems or portal systems or the general circulation, that is in getting to adjacent cells in a sort of slow and diffused way. For this I brought into the picture those cases where we have stroma in between, or call it ground substance. Such things do exist in certain cases. Your question whether this occurs in the central nervous system is at the moment not to be answered in the affirmative because we have no basis for it. The trouble is that you are right in supposing something of a transmitter type action which lasts a thousandth of a second and does not serve our purposes well. But we have other methods, the neuron is more versatile than the classic neurophysiologists needed to deal with.