TiPS - December
1992 [Vol. 131
429
chemical, mechanisms. To lump these two mechanisms into a single category, that of positive reinforcement, is an unwarranted oversimplification. Similar considerations apply specifically to the distinction between positive and negative reinforcement in drug-seeking behaviour, where these two conditions might invoive different neurochemical mechanisms and even a different therapeutic strategy. In conclusion, the definition of drug self-administration simply as a positive reinforcement mechanism, although technically correct, appears insufficient to describe the different processes involved in drug abuse. GAETANO DI CHIARA Department
of Toxicology, University
of
Cagliari, Viale A. Diaz 182, 09126 Caglirrri, Italy.
References 1 Stolennan, I. P. (1992) Trends Pharmacol. Sci. 13,170-176
2 Di Chiara, G. and North, R. A. (1992) Trends Pharmacol. Sci. 13, X35-193
Stolen-mmreplies Di Chiara offers an Interesting perspective on mechanisms of drug self-administration that contrasts with the position expressed in my TiPS review. My point was not that the mechanisms of reinforcement in the two cases discussed were identical but that the term ‘negative reinforcement’ is not appropriate for describing either of them. Instead, 1 maintain that negative reinforcement is a term more appropriately reserved for situations where prevention of drug effects constitutes the reinforcing event (i.e. avoidance of, or escape from, the administration of a drug). Like Di Chiara, I acknowledge diffenmces in neurochemical mechanisms and brain sites depending on whether drugs maintain self-administration behaviour through direct effects or indirectly through termination of withdrawal relief. Indeed, I go further and believe that several mechanisms may be involved even when only direct effects of reinforcement. mediate drugs These direct effects of drugs may be complex and multiple, including, but not limited to, euphoriant
subjective effects, termination of negative affective states (due to drug withdrawal or other reasons) and cognition and performance enhancements. In this schema, positive reinforcement is seen not as a unitary process, but instead as a unifying and operationally defined concept for behavioural changes that may be mediated through several mechanisms. I believe that the common assumption that drug self-administration can be classified accurately and
simply into cases of positive and negative reinforcement is erroneous and my position, rather than being an oversimplification, implies a need for a classification more rather than less complex than that proposed by Di Chiara. I look forward to seeing further debate on the issue. IANsroLEIWAN Institute of Psych&y,
De Crespigny Park,
London, UK SE5 SAF.
Tmsmitter concentration at the synapse
Unravellingthe paradox In a recent issue of TiPS, Samson and Harris’ included, in their article on ‘Neurobiology of alcohol abuse’, a section entitled ‘Drugs and synapses: puzzles and paradoxes’. But I do not think that there are really any paradoxes. They point out, for example, that in a ln~ solution there will be only one drug molecule for every 100 synaptic clefts. However, once the system as a whole has come to equilibrium the average concentration in the synaptic cleft is inevitably, nevertheless, 1 no. The actual moment-to-moment concentration will of course fluctuate, as in any small volume, but this does not alter the fact that, when averaged over time, the concentration in the cleft will be the same as anywhere else (and the length of time over which you have to average will be quite short on the time scale of most experiments). Therefore, if, for example, the equilibrium constant for binding is also ln~ then 50% occupancy will be achieved at equilibrium, and there is no need to postulate any mysterious accumulation at The restricted the synapse. volume of the synaptic cleft will of course slow down the rate at which equilibrium is achieved. It will also mean that, for example, any antagonist that dissociates from the receptor (e.g. when agonist is added) will transiently cause a disproportionately large rise in free antagonist concentration in the cleft, and this may have the effect of making the antagonist seem more potent (for brief agonist applications) than one might expect from its equilibrium constant, rather than being ineffective,
as the authors suggest. These phenomena are quite well understood and have been much discussed in the literature (for examples see Refs 24). Samson and Harris also suggest that it is also a puzzle that synaptic currents can be potentiated (e.g. by benzodiazepines) if the transmitter saturates the postsynaptic receptors. This question has been investigated in great detail in the case of anticholinesterases at the neuromuscular junction (for examples see Refs 5-7). The postsynaptic receptors may indeed approach saturation, but only over a very smalI area and for a very short time. This may be why anticholinesterases usually have a more prominent effect on the duration of postsynaptic currents than on their peak amplitude. But either way, the total charge passed is increased so the effectiveness of the postsynaptic current in producing depolarization is increased. Thus no problems arise as long as, when dealing with one or a few molecules, one thinks in terms of averages over time. In the case of a large collection of ion channels one can say that at any instant, 1% (say) of all channels is open, but a single channel is either fully open or fully shut - it is open for 1% of the time (on average). D. COLQDHODN Depattment of College London, WClE 6BT.
Phamacology, University Gower Street, London, UK
References 1 Samson, H. H. and Harris, R. R (1992) Trends Phamacol. Sci. 13,206-211
Q 1992.Elsevier Science Publishem Ltd CUW
TiPS - December 2 Waud, D. R. (1968) Pharmacol. Rev. 20, 49-88 3 Colquhoun, 0. and Ritchie, J. M. (1973) A9d. PhQTlQQCOk8,285-292 4 Katz, B. and Miledi, R. (1973) 1. Physiol. (Lo&.J 231,549-574 5 ibkell, H. C., Kuffler, S. W. and Yo&kami. 0. (1975) 1. Phvsiol. (Land.)
251,427& .
--
-
S. W. and Ywhikami. D. (1975) 1. Physiol. Kond.~ 251,465-482 7 PenneFather, P. and Quaat& 0. M. J. (1981) I. GeR. Pkysioi. 78.313-344 6 hffler.
HaMsandSamson
reply CoIquhoun raises several important points regading the interpretation of synaptic concentrations of drugs and quite corm&y points out that these issues have been considered by
pharmacologists in many previous articles. With regard to potentiation of synaptic currents by benzodiazepines, Colquhoun notes studies of inhibitors of acetylcholinesterases predict that benzodiazepines should increase the duration but not the amplitude of IPSPs. If this were always the case, there would indeed be no puzzle concerning synaptic actions of these drugs. However, in a number of studies12 it was found that benzodiazepines increase the amplitude of the postsynaptic response mediated by synaptic release of GABA. By contrast, when exogenous GABA is used, benzodiazepines do not alter the action of a maximally effective concentration of GABA but increase the apparent affinity of GABA for GABA* receptors-. Thus, we feel that there
How to estimate the total receptor concentration when the specific radioactivity of the ligand is unknown Stephane Swillens A computation method is presented allowing a direct determination of the total receptor concentration from a competitive displacement curve. Compared to fhe classical method based on the numerical processing of binding data, the proposed method has the advantage of being more accurate and of not requiring knowledge of the specific radioactivity of the ligand. An experimenter who wants to give a macroscopic description of the coupling between a pharmacological receptor and a specific ligand usually has to propose a binding model, to verify its validity, and to estimate the parameters defined in the model, such as for instance the equilibrium dissociation constant and the S. SmiUensis Ckrrrge’de Cows nt the Instihrt de Rechexhe Interdisciplinaire, UnivenitP
Libre de Bruxelles, Campus Erusme, bdt. C, route de Lennik 808, B 1070 Brussels, Belgium.
8
1992, Elsevim Science Publishem Ltd (UK)
number of binding sites. For that purpose, kinetic or equilibrium experiments are performed in which a radiolabelled ligand is incubated in the presence of the receptor. After the incubation, the bound fraction of the radioligand is separated from the free ligand molecules and counted. The data are then interpreted on the basis of a graphical linear representation, such as the Scatchard plot1 in the case of equilibrium data, or better, on the basis of a computer analysis by nonlinear regression*.
1992 [Vol. 131
is a contradiction between the assumption that synaptic release of GABA results in full occupancy of receptors and the observations that benzodiazepines can increase the amplitude of IPSPs but do not increase the action of a maximally effective concentration of GABA. R. A. HARRIS AND *H. H. SAMSON
Department of Phnrmaco~ogy, University of Colorado School of Medicine, Denver, CO and *Department of Psychiatry and Behavioral Sciences, University of Washington School of Medicine, Seattle, WA, USA.
References 1 Alger, B. E. and NicoB, R. A. (1982) 1. Physiol. (Land.) 328,105-123 2 Segal, M. and Barker, J. L. (1984)
J. Neurophysiol. 52,469-487 3 Hattori, K., Oomura, Y. and Akaike, N. (1986) Cell. Mol. NeurobioI. 6,307323 4 Yakushiji, T., Tokutomi, N. and Akaike, N. (1989) Neurosci. Res. 6, 309-320 5 Akaike, N., Oyama, Y. and Yakushiji, T. (1989) Brain Res. 504,293-296
In the past, most binding studies were aimed at characterizing the respective equilibrium dissociation constants of a series of ligands for a receptor type. More recently, the rapidly emerging techniques in the field of molecular biology have led to the cloning and sequencing of a number of receptors. It has thus become possible to produce a large amount of recombinant receptors in transfected cell lines. One of the questions addressed in those studies concerned the level of receptor expression. Binding experiments using a specific radioligand had thus to be performed in order to determine the number of receptors per cell. Some weeks ago, I was tackled by a colleague who was confronted with the following situation: he had expressed a known receptor in a cell line and wanted to determine the binding capacity of these cells for the specific hormone, but unfortunately, for some technical reasons, the specific radioactivity of the radiolabelled hormone was unknown. So, the question was: ‘Is it possible to estimate the maximal specific binding (E,,,) without knowing the specific radioactivity of the ligand?’ The immediate and intuitive answer I gave was negative, just because this maximal binding will be expressed in
1992 [Vol. 131
429
chemical, mechanisms. To lump these two mechanisms into a single category, that of positive reinforcement, is an unwarranted oversimplification. Similar considerations apply specifically to the distinction between positive and negative reinforcement in drug-seeking behaviour, where these two conditions might invoive different neurochemical mechanisms and even a different therapeutic strategy. In conclusion, the definition of drug self-administration simply as a positive reinforcement mechanism, although technically correct, appears insufficient to describe the different processes involved in drug abuse. GAETANO DI CHIARA Department
of Toxicology, University
of
Cagliari, Viale A. Diaz 182, 09126 Caglirrri, Italy.
References 1 Stolennan, I. P. (1992) Trends Pharmacol. Sci. 13,170-176
2 Di Chiara, G. and North, R. A. (1992) Trends Pharmacol. Sci. 13, X35-193
Stolen-mmreplies Di Chiara offers an Interesting perspective on mechanisms of drug self-administration that contrasts with the position expressed in my TiPS review. My point was not that the mechanisms of reinforcement in the two cases discussed were identical but that the term ‘negative reinforcement’ is not appropriate for describing either of them. Instead, 1 maintain that negative reinforcement is a term more appropriately reserved for situations where prevention of drug effects constitutes the reinforcing event (i.e. avoidance of, or escape from, the administration of a drug). Like Di Chiara, I acknowledge diffenmces in neurochemical mechanisms and brain sites depending on whether drugs maintain self-administration behaviour through direct effects or indirectly through termination of withdrawal relief. Indeed, I go further and believe that several mechanisms may be involved even when only direct effects of reinforcement. mediate drugs These direct effects of drugs may be complex and multiple, including, but not limited to, euphoriant
subjective effects, termination of negative affective states (due to drug withdrawal or other reasons) and cognition and performance enhancements. In this schema, positive reinforcement is seen not as a unitary process, but instead as a unifying and operationally defined concept for behavioural changes that may be mediated through several mechanisms. I believe that the common assumption that drug self-administration can be classified accurately and
simply into cases of positive and negative reinforcement is erroneous and my position, rather than being an oversimplification, implies a need for a classification more rather than less complex than that proposed by Di Chiara. I look forward to seeing further debate on the issue. IANsroLEIWAN Institute of Psych&y,
De Crespigny Park,
London, UK SE5 SAF.
Tmsmitter concentration at the synapse
Unravellingthe paradox In a recent issue of TiPS, Samson and Harris’ included, in their article on ‘Neurobiology of alcohol abuse’, a section entitled ‘Drugs and synapses: puzzles and paradoxes’. But I do not think that there are really any paradoxes. They point out, for example, that in a ln~ solution there will be only one drug molecule for every 100 synaptic clefts. However, once the system as a whole has come to equilibrium the average concentration in the synaptic cleft is inevitably, nevertheless, 1 no. The actual moment-to-moment concentration will of course fluctuate, as in any small volume, but this does not alter the fact that, when averaged over time, the concentration in the cleft will be the same as anywhere else (and the length of time over which you have to average will be quite short on the time scale of most experiments). Therefore, if, for example, the equilibrium constant for binding is also ln~ then 50% occupancy will be achieved at equilibrium, and there is no need to postulate any mysterious accumulation at The restricted the synapse. volume of the synaptic cleft will of course slow down the rate at which equilibrium is achieved. It will also mean that, for example, any antagonist that dissociates from the receptor (e.g. when agonist is added) will transiently cause a disproportionately large rise in free antagonist concentration in the cleft, and this may have the effect of making the antagonist seem more potent (for brief agonist applications) than one might expect from its equilibrium constant, rather than being ineffective,
as the authors suggest. These phenomena are quite well understood and have been much discussed in the literature (for examples see Refs 24). Samson and Harris also suggest that it is also a puzzle that synaptic currents can be potentiated (e.g. by benzodiazepines) if the transmitter saturates the postsynaptic receptors. This question has been investigated in great detail in the case of anticholinesterases at the neuromuscular junction (for examples see Refs 5-7). The postsynaptic receptors may indeed approach saturation, but only over a very smalI area and for a very short time. This may be why anticholinesterases usually have a more prominent effect on the duration of postsynaptic currents than on their peak amplitude. But either way, the total charge passed is increased so the effectiveness of the postsynaptic current in producing depolarization is increased. Thus no problems arise as long as, when dealing with one or a few molecules, one thinks in terms of averages over time. In the case of a large collection of ion channels one can say that at any instant, 1% (say) of all channels is open, but a single channel is either fully open or fully shut - it is open for 1% of the time (on average). D. COLQDHODN Depattment of College London, WClE 6BT.
Phamacology, University Gower Street, London, UK
References 1 Samson, H. H. and Harris, R. R (1992) Trends Phamacol. Sci. 13,206-211
Q 1992.Elsevier Science Publishem Ltd CUW
TiPS - December 2 Waud, D. R. (1968) Pharmacol. Rev. 20, 49-88 3 Colquhoun, 0. and Ritchie, J. M. (1973) A9d. PhQTlQQCOk8,285-292 4 Katz, B. and Miledi, R. (1973) 1. Physiol. (Lo&.J 231,549-574 5 ibkell, H. C., Kuffler, S. W. and Yo&kami. 0. (1975) 1. Phvsiol. (Land.)
251,427& .
--
-
S. W. and Ywhikami. D. (1975) 1. Physiol. Kond.~ 251,465-482 7 PenneFather, P. and Quaat& 0. M. J. (1981) I. GeR. Pkysioi. 78.313-344 6 hffler.
HaMsandSamson
reply CoIquhoun raises several important points regading the interpretation of synaptic concentrations of drugs and quite corm&y points out that these issues have been considered by
pharmacologists in many previous articles. With regard to potentiation of synaptic currents by benzodiazepines, Colquhoun notes studies of inhibitors of acetylcholinesterases predict that benzodiazepines should increase the duration but not the amplitude of IPSPs. If this were always the case, there would indeed be no puzzle concerning synaptic actions of these drugs. However, in a number of studies12 it was found that benzodiazepines increase the amplitude of the postsynaptic response mediated by synaptic release of GABA. By contrast, when exogenous GABA is used, benzodiazepines do not alter the action of a maximally effective concentration of GABA but increase the apparent affinity of GABA for GABA* receptors-. Thus, we feel that there
How to estimate the total receptor concentration when the specific radioactivity of the ligand is unknown Stephane Swillens A computation method is presented allowing a direct determination of the total receptor concentration from a competitive displacement curve. Compared to fhe classical method based on the numerical processing of binding data, the proposed method has the advantage of being more accurate and of not requiring knowledge of the specific radioactivity of the ligand. An experimenter who wants to give a macroscopic description of the coupling between a pharmacological receptor and a specific ligand usually has to propose a binding model, to verify its validity, and to estimate the parameters defined in the model, such as for instance the equilibrium dissociation constant and the S. SmiUensis Ckrrrge’de Cows nt the Instihrt de Rechexhe Interdisciplinaire, UnivenitP
Libre de Bruxelles, Campus Erusme, bdt. C, route de Lennik 808, B 1070 Brussels, Belgium.
8
1992, Elsevim Science Publishem Ltd (UK)
number of binding sites. For that purpose, kinetic or equilibrium experiments are performed in which a radiolabelled ligand is incubated in the presence of the receptor. After the incubation, the bound fraction of the radioligand is separated from the free ligand molecules and counted. The data are then interpreted on the basis of a graphical linear representation, such as the Scatchard plot1 in the case of equilibrium data, or better, on the basis of a computer analysis by nonlinear regression*.
1992 [Vol. 131
is a contradiction between the assumption that synaptic release of GABA results in full occupancy of receptors and the observations that benzodiazepines can increase the amplitude of IPSPs but do not increase the action of a maximally effective concentration of GABA. R. A. HARRIS AND *H. H. SAMSON
Department of Phnrmaco~ogy, University of Colorado School of Medicine, Denver, CO and *Department of Psychiatry and Behavioral Sciences, University of Washington School of Medicine, Seattle, WA, USA.
References 1 Alger, B. E. and NicoB, R. A. (1982) 1. Physiol. (Land.) 328,105-123 2 Segal, M. and Barker, J. L. (1984)
J. Neurophysiol. 52,469-487 3 Hattori, K., Oomura, Y. and Akaike, N. (1986) Cell. Mol. NeurobioI. 6,307323 4 Yakushiji, T., Tokutomi, N. and Akaike, N. (1989) Neurosci. Res. 6, 309-320 5 Akaike, N., Oyama, Y. and Yakushiji, T. (1989) Brain Res. 504,293-296
In the past, most binding studies were aimed at characterizing the respective equilibrium dissociation constants of a series of ligands for a receptor type. More recently, the rapidly emerging techniques in the field of molecular biology have led to the cloning and sequencing of a number of receptors. It has thus become possible to produce a large amount of recombinant receptors in transfected cell lines. One of the questions addressed in those studies concerned the level of receptor expression. Binding experiments using a specific radioligand had thus to be performed in order to determine the number of receptors per cell. Some weeks ago, I was tackled by a colleague who was confronted with the following situation: he had expressed a known receptor in a cell line and wanted to determine the binding capacity of these cells for the specific hormone, but unfortunately, for some technical reasons, the specific radioactivity of the radiolabelled hormone was unknown. So, the question was: ‘Is it possible to estimate the maximal specific binding (E,,,) without knowing the specific radioactivity of the ligand?’ The immediate and intuitive answer I gave was negative, just because this maximal binding will be expressed in
