GABA: history and perspectives1 ERNSTFLOREY Fakultdt fur Biologic, Universitiir Konstanz, 0775 Konstanz I , Germany
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Received July 16, 1990 F~IPEY E. , 1991. GABA: history and prspectives. Can. J. Physiol. Pharmacol. 69: 1049- 1056. In 1957, factor I, a brain agent I had discovered earlier, was chemically identified as GABA in a collaboration between myself and A1va Bazemore at the MontrCal Neurological Institute (MNI) in the Neurochemistry Laboratory then headed by K. A. C. Elliott. A personally biased excursion into the history of neurobiology illuminates the development of methods and concepts that led to this event, and recounts the early days at the MNI, when Hugh McLennan and I applied factor I to the exposed surface OF the spinal cord and to sympathetic ganglia of cats and rabbits. It also tells sf earlier studies at Graz, Naples, and elsewhere that prompted the experiments at the California Institute of Technology in which factor K was discovered as the agent in nerve extracts causing inhibition of isolated crayfish stretch receptor neurons, and in which it was found that this inhibition could be prevented by picrotoxin. There was justified doubt that GABA is indeed the transmitter substance of inhibitory neurones. Later studies, however, resolved the controversy. The functional role of GABA in brain and spinal cord and its mechanism of action are still far from being filly understood. Special problems are the extent and significance of spontaneous quanta1 and nonquantal release, the hnctional role and the mechanism of excitatory actions of GABA, its release from glial cells, and the energetics of its metabolic turnover. Key words: factor I, GABA, glia, convulsants, inhibition. F~REY E., 1991. GABA: history and perspectives. Can. J. Physiol. Pharmacd. 69 : 1049- 1056. En 1957, lors d'un travail conjoint a 1'Institut neurologique de Montrbl (INM), dans le Laboratoire de neurochimie dirige alors par K. A. C. Elliott, Alva Bazemore et moi-mCme avons chimiquement identifib le GABA cesmme dtant 1e facteur 1, une substance cCrCbrale que j'avais dkouverte antkrieurement. Une retour i l'histoire de la neurobiologie fait la lumiere sur le dCveloppement des mtthodes et concepts qui ont men6 i cette dCcouverte et rappelle les dCbuts h I'INM, lorsque Hugh McLennan et moi-mCme avons applique le facteur I h la surface exposCe de la moelle Cpin5re et aux ganglions sympathiques de chats et de lapins. On retient aussi les Ctudes 2 Graz, Naples et autres endroits qui ont precCdC les expkriences au California Institute of Technology, ou le facteur I a CtC identifiC comme la substance provoquant l'inhitaition des neurones des rCcepteurs d9hirementisolCs de l'bcrevisse, et oh on a constat6 que la picrotoxine p~uvaitempCcher cette inhibition. On csoyait alors que le GABA pouvait Ctre la substance mCdiatrice des neurones inhibiteurs, ce que des Ctudes ultCrieures ont permis de confirmer. Le r61e fonctionnel de GABA dans le cerveau et la moelle Cpiniere et son mdcanisme d'action sont encore loin d'etse totalement compris, notamment 19amplitudeet l'importance de la 1ibCration spontanCe non quantique et quantique, le r61e fonctionnel et le mkcanisme des actions excitatrices de GABA, sa 1ibCration des cellules gliales, et le volet Cnergdtique de son renouvellement mCtabolique. Mots cl&s : facteur I, GABA, cellule gliale, convulsivants, inhibition. [Traduit par la rbdaction]
Introduction As we get older, the world around us seems to get younger. Our colleagues now are mostly young fellows, and our chosen field of science rushes on with ever-increasing speed. However, our past stays with us, and what we did 30 or 40 years ago is still alive in our memories. The problems we worked on keep nagging at us as we realize that the answers we sought are by no means final. 'The little discoveries we made still demand further explorations. Our own past is still very much alive, and we realize that it is not really our own private creation, or our private possession. As scientists we have accepted the framework of thought that our teachers, and their teachers, had prepared for us. And where we succeeded, our endeavours became part of the mainstream of science because they fitted into its accepted framework. As we get older, we become more and more aware that science is not only a future but that it is also a memory of thoughts and of ideas reaching far back into the past. When viewed in the historical context, physiology is a relatively recent part of science. 'The physiology of the nervous system has a history of more than 28QM)years. Yet we are still at the 'This paper was presented at the symposium to honour Dr. Hugh McLennan, Amino Acids as Neurotransmitters, held at the University of British Columbia, Vancouver, B.C., May 28 -29, 1990, and has undergone the Journal's usual peer review. Printed in Canada i lrnprimi au Canada
beginning as we ponder the dual aspect of our existence, the world around us and the world within, the relationship of physical processes within the nervous system, and the experiences of the mind. The inner mental life is not only the representation of the ever-changing features of the outside world extracted by our sense organs and elaborated in our brains, but it is also the inner process s f rational, and sometimes irrational, thought, of feelings and emotions, and of imagination, contemplation, and the preparation of our overt behaviour. As we review the history of our science we realize that its prime motivation has always been the search for an explanation and understanding of the relationship between our inner and outer worlds. From ancient Greece, from A l h a i o n , Hippocrates, Plato, Aristotle, Herophilos, Erasistratos, and Galenos, through Vesalius, Descartes, and Willis, to Haller , Soemmering, Gall, Broca, Retzius, and finally to Papes, Ranson-Clark, and Rasmussen, there runs a continuous line of explorations of the possibility that the anatomy of the nervous system might be the key to an understanding of the role of the nervous system and its chief organ, the brain, as the mediator between the physical and the mental, between body and mind. Physiologists had to wait for the technological advances that would make a physical investigation of the functioning of the nervous system possible. Electrophysiology thus assumed a dominant role in the 19th century, while a chemical approach developed even more slowly, in spite of the inducements of
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pharmacological experience. Neurochemistry matured only as the present century reached its midpoint. Canadian neurochemists played a major role in this: witness the first textbook Neurochemistry (Elliott et al. 1955). No wonder that it was in Canada that the GABA story came into focus. Hugh M c h n n a n whom we honor at this conference had no small part in this. In the following sketch I will attempt a review of the history that led to the discovery that y-aminobutyric acid (GABA) might be a transmitter substance of inhibitory neurons, and H will explain the ensuing doubts in such an identification. I will also show that the GABA story is by no means finished.
From excitation to inhibition Albrecht von Haller (1708 - 1777) focused attention on the fundamental characteristic of nerves: excitability. With the research of Emil Du Bois-Weymond (18 18- 1896) began the recognition that the electrical signal we now refer to as the nerve impulse is the faanciamental event in the transmission of excitation from sense organ to spinal cord and brain, and from spinal cord and brain to the muscles. By the end s f the last century, the concept of inhibition as a complementary process in the central nervous system became established. Already in 1845 (Weber and Weber 1845), the German physiologists Ernst Heinrich Weber (1795- 1878) and his brother Friedrich Wilhelm W e k r (1806 - 1871) discovered the newe-mediated inhibition of the mammalian heart. Ivan Pavlov (1849- 1936) described nerve-mediated inhibition of muscle contraction in a clam (Pavlov 1885). Wilhelm Biedermann (1852 - 1929), the physiologist at the University s f Jena, discovered that in crayfish, the leg muscles receive an inhibitory innervation (Biedermann 1887). Eater, the Dutch physiologist Cornelis Adrianus Gerrit Wiersma (1905 - 1979) elucidated the anatomy and functional significance of these inhibitory neurons Wiersma 1933). The application of the microelectrode and the necessary cathode-follower amplifiers in Gerard's laboratory in the 5th decade of our century (Graham and Gerard 1946) made possible the discovery of the ionic mechanism involved in the inhibitory synaptic transmission in the spinal cord of cats. Already 4 years later, Brock et id. (1952) reported inhibitory postsynaptic potentials in spinal motoneurons of the cat, and by 1955 the use of double-barrelled microelectrodes established the theory that the inhibitory postsynaptic potential is due to an increase of chloride conductance (Cmmbs et al. 1955). In the same year, Muffler and Eyzaguirre (1955) described inhibitory postsynaptic potentials in the large isolated stretch receptor neurons s f crayfish, which had been described 4 years earlier by Alexandrowicz (195 1). Physiological data together with extensive anatomical and electrophysiological analyses of the nervous pathways had now provided ample evidence for the reality s f inhibitory processes in the nervous system and for their importance. Within another decade it emerged that within the mammalian central nervous system inhibitory neurons may outnumber excitatory ones. However, at this point I am getting ahead of my story. In 1955, John Carew E c l e s delivered three invitational lectures at the Johns Hopkns University in Baltimore where Stephen Kuffler (19 13- 1980) and his Chilean colleague Carlos Eyzaguirre had studied crustacean stretch receptor neurons. Their now classic pagers of 1955 (Eyzaguirre and
Muffler 1955a, 195567) in the Journal of General Physiology are milestones in the physiology of sensory cells as well as the physiology of synapses. Eccles' lectures resulted in his famous book The physds&ogysf nerve cells (Eccles 1957), which combines the evidence from both cat and crayfish in an epochmaking new synthesis resulting in a theory of the functioning of nervous systems that dominated neurophysiology for years to come.
Is GABA the transmitter? Chemical synaptic transmission had been firmly established by the mid-1950s. The transmitter substance of the so important inhibitory neurons, however, had still escaped detection. Today, nearly four decades later, we are confident that GABA and its hornologue glycine are the transmitter substances of most inhibitory neurones. In retrospect, it seems incredible that it should have taken no more than 2 years between the publication of incontrovertible proof that transmission at inhibitory synapses is chemically mediated and the identification in 1957 of GABA as the substance most likely to be the transmitter substance at inhibitory synapses (Bazemore et al. 1957). Indeed, rigorous testing of the substance by Curtis et al. (1959) revealed that in contrast with the expectations of Bazernasre et al. (1957), GABA did not hlfill the criteria of a transmitter substance. A chief problem was the fact that the action of GABA was not blocked by strychnine, the alkaloid that was already known to block the action of the inhibitory terminals on motoneurons. It took another decade of research in which many of the colleagues present at this meeting participated to provide evidence that the transmitter substance blocked by strychnine is identical with glycine, and that GABA is the transmitter substance of another class of inhibitory neurons whose action is indeed blocked by picrotoxin. Our honored friend Hugh McLennan has recorded the evidence in the second edition of his well-known book Synaptic tran~rnis~ion, which was published in 1970. Today, GABA is thought to be the prevalent transmitter substance of the vertebrate central nervous system and of our own brain. Compared with the steady-state m o u n t s of the other transmitters, the GABA content of the mammalian brain is indeed astonishing: up to I mglg wet weight of brain tissue (e.g ., Roberts 1962)0 Its unique, and apparently exclusive, association with the central nervous system had suggested a special role in the functioning of brain and spinal cord, possibly a metabolic function (Roberts 1962). How did this amino acid, GABA, become connected with chemical transmission at inhibitory synapses? This is an interesting story that demonstrates how closely the history of science is linked with the personal history of the scientists whose work, after all, has created this science. The scientific discoveries are even related to the political history since every scientist is part, and oken victim, of that history.
Setting the stage As I look back at the unfolding GABA story, I see it from my own personal perspective and I hope you will forgive my undenied personal bias. My story begins with my student days at the University of Graz in Austria. My physiology teacher and thesis supervisor was Karl Umrath (1899- 1985) who had been trained at the Universities of Prague and Jena. The physiologist at Jena was Wilhelm Biedermann (1852 - 1929) who had discovered the inhibitory innervation of crayfish skeletal
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muscle. We will see later on how important Biedermann was to the GABA story. Umrath moved to Graz, where he later became Assistant to Professor Otto Loewi (1873 - 1961) in the Department of Pharmacology, and then assumed a position of Associate Professor in the Zoology Department of the same university. In the 1930s, Umrath became a well-known biophysicist with a strong mahemtical bend, but at heart he was a comparative physiologist and this is how I came to know him. Umrath suggested that I work out the mechanism of the central action of convulsant drugs, assuming that such substances as strychnine, metrazol, and picrotoxin might have an action analogous to that of physostigmine (eserine), namely, the inhibition of an enzyme that degrades the elusive transmitter substance of sensory neurons. With another former assistant of Otto Loewi7s, Horst Hellauer, Umrath had made use of the so-called axon reflex (it results in a local vasodilatation in the skin when the skin is mechanically irritated). The physiological preparation was the denervated ear of albino rabbits. Subcutaneous injection of extracts of dorsal spinal roots caused conspicuous reddening, which did not occur when extracts of ventral roots were injected. For reasons I cannot fathom today, H did not use this preparation; but with the blessing of Umrath and of Karl von Frisch (1 886- 1982), I travelled to the famous Stazione Zoologica at Naples to embark on a comparative investigation of the effects of convulsants in a vast number of different animal species. I had the benefit of being assigned to the large laboratory suite of the Belgian pharmacologist, Z. M. Bacq (1903 - 1983), a most enthusiastic and temperamental experimenter from whom I acquired much theoretical and practical knowledge of pharmacology. When I reviewed my data in the context of zoological systematics (Florey 1951), I realized that none of the arthropod species responded to strychnine but that they were extremely sensitive to picrotoxin; with molluscan species it was the other way around. Vertebrates and their chordate relatives, the chaetognaths, echinoderms, and tunicates, responded with convulsions when injected with either drug. Although H developed another promising pharmacological assay method, it was not possible to find any evidence that the convulsants had anything to do with sensory neurons. With a postdoctoral fellowship I moved to Gottingen to learn the new techniques of electrophysiology in the laboratory of Hans-Jochen Autrum. A Fullbright grant and a Hixon Fellowship then brought me to the laboratory of C. A. G. Wiersma at the California Institute of Technology in Pasadena. A few weeks after my arrival there in the summer of 1951, Wiersma received a reprint from the great comparative neuroanatomist J. S. Alexandrowicz (1886- 1970) of the Plymouth Marine Laboratory. This paper reported the discovery of muscle receptor organs in the abdominal extensor muscles of lobsters, which consist of two muscle strands and two large neurons whose cell bodies are placed near the muscle strands. The presence of an efferent innervation suggested a role similar to that of the mammalian muscle spindles except that the sensory neurons also received an innervation. Alexandrowicz speculated that these stretch receptor neurons receive both an excitatory and an inhibitory synaptic input. Wiersma immediately realized the importance of this discovery for physiology. He convinced his Ph.D. student Edwin Furshpan and me that we ought to devote all our energies to develop a physiological stretch receptor preparation suitable for physiological experiments. Secretly, I hoped to use this preparation to carry out phar-
macological experiments, since such a preparation would offer an unusual chance to study the action of drugs on synaptic transmission with identified nerve cells. At that time, chemical synaptic transmission was not yet generally accepted; Wiersma certainly did not like it. It was only after he took a sabbatical leave of absence for a return to his beloved Holland that I seized the chance to test nerve extracts and drugs on stretch receptor neurons. Ed Furshpan developed a very nice semiisolated preparation but then decided to change his research topic and to turn to nerve-muscle preparations. This left me in full possession of a lovely test system. In my newly acquired ancient automobile, a 1934 Chevrolet, I travelled frequently to a horse butcher in Los Angeles to obtain spinal cord, brain, and nerve roots from freshly killed horses. With much advice from CalTech chemists, I attempted the purification of a substance, or mixture of substances, responsible for a curious action of certain extracts on the sensory cells: such extracts inhibited the firing of the receptor neurons and made them unresponsive to stretch. The effect could readily be blocked by picrotoxin but not by strychnine. Considerable purification of the inhibitory agent (1 later called it "factor I") was achieved, but my nonrenewable visa ran out and, regretfully, I had to return to Austria. I was not too unhappy, though, as I was now able to get married and to take my wife Elisabeth, another Umrath pupil, with me to the Zoology Department of Wiirzburg University. Autrum had become chairman of the department there and invited us to work in his laboratory. John Welsh, then chairman of the Biology Department of Harvard University, had provided me with an oscilloscope, amplifier, and stimulator, and Autrum let us use a research microscope. We were thus able to embark on a further study of crayfish stretch receptor neurons and I finished the manuscript on my work in Pasadena; it was published in 1953 (Florey 1953). At that time an invitation came from K. A. C. Elliott, head of the Donner Laboratory of Neurochemistry at the Montrbal Neurological Institute (simply referred to as the MNI), to continue work on the inhibitory factor. Elisabeth was given an opportunity to work with George (Jerzy) Olszewski (1913 - 1964) in the Department of Neuroanatomy in the same MNI. We applied for an immigration visa and a few months later we embarked on a freighter that took us to Montreal where we arrived amidst spectacular ice flows in the spring of 1954. Another postdoctoral fellow had just joined Elliott. A former McGill graduate, he had returned from the laboratory of C . L. Brown (1903 - 1971) in London where he acquired great skills in operating on cats and rabbits. He was none other than Hugh McLennan. However, before I continue my personal account, let me return once more to history.
The Biedermann tradition and the Plymouth csnnection Wilhelm Biedermann, whom I mentioned earlier, was not only the teacher of my own teacher Umrath, he also taught Alexandrowicz and the Dutch zoophysiologist Hemann Jacques J o r h (1877 - 1943) who later b e m e chairman of the Zoology Department of Utrecht University and who took on C. A. G. Wiersma as his assistant, giving him the task of pursuing nerve -muscle physiology in crayfish. The Biedermann tradition sparked Wiersma7s reception of Alexandrowicz' discovery. But how did Stephen Kuffler become interested in working with crayfish stretch receptor organs in a laboratory that was devoted to ophthalmological research?
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Mere our story touches on political history. When the National Socialists took over Germany, Bernard Katz, who received his doctorate from Eeipzig University in 1935, sought refuge in England. The famous A. M. Mill (1886-1977) promised to help but at the moment had no vacant position. Mill had a house near Plymouth and he was a member of the governing board sf the Plymouth Marine Laboratory. He suggested that Katz spend some time there until a place could be found for him in bndon. At Plymouth, it was Carl Frederick Ak1 Pantin (1899 - 1951) who introduced Katz to crustacean nerve muscle physiology. From 1922- 1929, Pantin had been resident physiologist at the Plymouth Marine Laboratory, and now that he had become Professor of Zoology at Cambridge, he was still a frequent visitor. Katz later emigrated to Australia and worked there in Ewles' laboratory where another refugee from Germany had found asylum: Stephen Kuffler (19131980). Katz passed on his knowledge of crustacean nervemuscle physiology to Kuffler, and after the war they did important research on synaptic transmission in the crab and crayfish nerve -muscle preparations. Kuffler thus knew the advantages of working with crayfish through the '6Plymouth connection," and when he learned of the stretch receptors from another Plymouth researcher, Alexandrowicz, he was well prepared. In the early 1950s, Kuffler started a research program to explore the advantages of the stretch receptor neurons Alexandrowicz had discovered. He invited me and my wife to join his laboratory, but we preferred to stay in Montreal. We did, however, enjoy a visit with Stephen Kuffler and Carlos Eyzaguirre in Baltimore in 1955; the result was the decision to publish our paper (Florey and FIorey 1955) on the microanatomy of crayfish stretch receptor neurons in the same issue of the Journal of General Physiology in which the three now classical papers of Kuffler and Eyzaguirre appeared in the following year. Montreal, in 1954, was a different place from what it is now. Wilder Benfield ( 1891- 1970), George OlszewsG, and Allan Elliott, so active at that time, are no longer with us, and most of the fellows we knew there have long since moved to other places. Many have become famous, among them David Hubel whom we knew as a recorder playing intern, who later joined Harvard University and received the Nobel Prize for his research on the visual system which Stephen Kuffler had also explored much earlier.
Research on factor I Hugh McLennan was keen to apply his new knowledge of neurophysiology and we soon discovered that our interests complemented each other very nicely indeed. We set up for experiments on stretch receptors to test for factor I activity and for experiments on cats to test the effect of purified fractions of nerve extracts on spinal reflexes and on synaptic transmission in sympathetic ganglia of cats and rabbits. Figure B illustrates our collaboration. In 1955 two of our papers appeared in the Journal of Bhysiology in which we reported that purified factor I inhibited the monosynaptic knee-jerk reflex when topically applied to the exposed spinal cord of decerebrated cats. This effect was prevented by strychnine. We also found that factor I inhibited synaptic transmission in the stellate and the inferior mesenteric ganglia sf cat and rabbit, while no effect was seen in the superior cervical ganglion. Curiously, we did not test the effect sf strychnine or picrotoxin in the sympathetic ganglia.
FIG. 1. Hugh McLennan (left) and Ernst FBorey at the Montrdal Neurdogical Institute in 1955, preparing a decerebrated cat for topical application of factor I to the exposed spinal cord.
Rather courageously we suggested that "factor I may be the transmitter substance of certain inhibitory neurones." A sample of our factor I preparation was sent to Eccles' laboratory and tested by David Curtis, John Eccles, and Rose M. Eccles who applied it to the spinal cord of cats by intra-arterial injection. It was found to be ineffective (Curtis et aB. 1958).
From factor I to GABA Meanwhile, Elliott secured the assistance of Merck Bs Co. at Rahway, New Jersey. They sent a chemist, Alva Bazemore, and offered us the facilities of their Montreal plant. A1 Bazemore and I spent many exciting hours performing large-scale extraction and purification of factor I, starting with more than 100 lb (1 lb = 0.454 kg) of beef brain with an extraction with 450 L of acetone, not to mention the other solvents we employed. Several different extraction steps and column chromatographic separation resulted in 420 mg of a crystalline material. If the entire extract had been purified, a total of 513 mg would have resulted. Further fractionation of 100 mg of this material by recrystallization yielded 18 mg of rhomboid crystals of homogeneous melting point. This and other data indicated that the substance is identical with GABA (Bazemore et al. 1957). The material co-chromtographed with authentic GABA. Both substances were similarly effective when tested on the stretch receptor neuron. As I now recalculate the results, it turns out that the isolation procedure yielded 11.78 mg GABA/ kg of original brain tissue, or 1.78 pglg of fresh brain tissue, certainly a small yield considering that the inhibitory effect of a fresh aqueous extract of 1 g of beef brain is equal to that of about 200 pg of GABA (see data of Florey and Florey 1958). Doubts A number of other discrepancies, such as the fact that the action of GABA on spinal motoneurons was not blocked by strychnine (as shown by Curtis et al. 1959), raised doubts that GABA was a transmitter substance. David Curtis in Australia had developed a most powerful tool, a multibarrelled assembly of microelectrodes that could be used for iontophoresis of several drug solutions to the immediate vicinity sf the site
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l o w (a) GABA
k2w (b) GABA
(0
high GABA
ml-
FIG. 2. The effect of low M) and high (lW4 M) concentrations of GABA ( a , b , c) and baclofen (d; los5 M) on the membrane potential and input resistance s f the slowly adapting stretch receptor neuron s f the crayfish (Orconectes kirnosus). Upper channel: current calibration and current signals; lower channel: membrane potential and action potentials. The drug application begins at the start of the slow recording speed. In Fig. 2b the neuron begins to fire. Note the transient excitation in Fig. 2c as the GABA concentration rises. Unbuffered saline.
from which an intracellular electrode recorded changes of membrane potential. Curtis and co-workers demonstrated further differences between the actions of GABA and those of the natural transmitter on spinal motoneurones, and in 1970 Hugh McLennan categorically stated "the rejection of GABA as a transmitter in the spinal cord remains" (McLennan 1970, p. 122). It is not necessary to go into further details. Suffice it to say that in the ensuing years it became evident that spinal inhibitory neurons do not produce GABA but another transmitter substance which, in all likelihood, is identical to glycine (Werman et al. 1968). In the brain, however, the predominant transmitter substance is indeed GABA. Puzzles remained concerning the role of GABA as a transmitter substance in crustaceans. GABA and the entire chemical machinery for its production and degradation were found to be present in crustacean nerve tissue and, indeed, nearly exclusively in axons of the inhibitory neurons present in peripheral nerve (e.g., Kravitz et d. 1963; Kravitz and Potter 196%;Sorenson 1973), but the inhibitory effect of extracted peripheral nerves (crustaceans) was very much greater than that accounted for by their GABA content (Florey and Chapman 1961). It was very puzzling that an extract containing
some 20 pg of GABA was so effective that it would have had to contain up to 5 mg of GABA if this substance were to account for the effectiveness of the extract. My colleague Bernd Koidl and I finally discovered that the effect of GABA could be mimicked by a mixture of amino acids, notably glutamate, aspartate, glycine, and taurine (Koidl and Florey 197%). Indeed, no GABA needs to be present in the mixture. What is more surprising is that the inhibitory effect (inhibition of impulse discharge in stretch receptor neurons and of contractile activity of the hindgut) can be blocked by picrotoxin. It would be most interesting now to investigate the nature of the picrotoxin-sensitive receptor responding to these amino acids. We have evidence that the GABA receptor of crayfish stretch receptor neurons differs from that of mammalian neurons in that the affinity for GABA is not altered by benzodiazepines (E. Florey and M . Rathmayer, unpublished).
Excitatory actions of GABA For several years now we have been studying an excitatory action of GABA on stretch receptor neurones. When this amino acid is applied in lower concentrations than those required to cause inhibition, it deplarizes the membrane and
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Astacus astacus
GABA x 1O - ~ M
Orconectes limosus
GABA XIO-~M
FIG.3. Slowly adapting stretch receptor neurons of two species of crayfish: dose-response curves of SABA. Upper panels: effect on input resistance (R,); lower panels: effect on membrane potential (Em).Number of experiments in parentheses. Unbuffered saline.
increases the membrane resistance. Examples and data are shown in Figs. 2 and 3. Dose-effect curves demonstrate the curious fact that there is an intermediate GABA concentration at which neither membrane potential nor membrane resistance are affected. The excitatory action is pH dependent. It occurs when unbuffered saline is used, but it disappears when the medium is buffered to pH 4 or higher. Both the excitatory and the inhibitory effects of GABA can be duplicated by muscimol, but this agonist is 5 to 10 times more potent than GABA so that inhibition occurs with concentrations down to M and excitation with concentrations as low as lW7 M. Picrotoxin converted the inhibitory effect of GABA and of muscimoI to an excitatory one; it seldom affected the excitatory actions of How GABA concentrations. Baclofen, a known GABAB agonist, when applied in concentrations of 4 X lo-" 4 X 1W6 M caused depolarization accompanied by a decrease of membrane resistance up to 30%- This effect was either not blocked or potentiated by picrotoxin. Bicuculine preferentially
reduced the excitatory actions of GABA; it usually enhanced the excitatory actions of baclofen. These findings imply that the excitatory actions seen with application of baclofen, GABA, and muscimol are not due to activation of a GABAB-typ receptor. I mention these results here because they show that the GABA story still has unexpected ramifications and leads to puzzling mysteries.
The role of glial cells hat me conclude by pointing out that recent evidence from several laboratories indicates that glial cells take up and release GABA (for references see the recent review by Erd6 and Wolff 1990). Only a very narrow gap separates glia and satellite cells from the neuronal surface, and even isolated neurones Hike the stretch receptor cells of crayfish carry an elaborate envelope of glial elements. It is not unreasonable, therefore, to suggest that amino acids and drugs act not only on the neuronal membrane but may indirectly affect the neu-
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FLOREY
ron through an action on the glia. There are 10 times as many glial cells in the mammalian brain than there are neurons. Surely, these cells must have considerable importance for brain function. The GABA content of nerve tissue may, to a major part, represent glial GABA. We have good evidence that GABA is continuously being released from nerve terminals; it may also be released from glial cells in a nonquantal fashion. Considering how narrow the extracellular space is between the cells, fluctuations of extracellular GABA concentrations can be expected to influence cell behaviour. The neuronal circuits we hear so much about may well be a fiction. Neurons are imbedded in glia and so are their circuits. The functioning of these circuits may well depend on the modulating influence of these glial cells. Perhaps, the GABA story will move in the direction of an elucidation of these relationships. The nervous system, certainly, is not an electrical apparatus as the artificial intelligence people want us to believe; it is a complex chemical machine, and much of the decisive chemistry takes place at the synapses. Synapses, however, are not all there is to a nervous system; they are simply the regulators of neuronal activity which, we grant that, can indeed be defined in terms of electrophysiology. But this neuronal activity, like that of the synapses, is also determined by the immediate environment (extracellular matter, extracellular fluid) and by the action of glial cells. We know that glia responds to neuronal activity, absorbing released ions and transmitter substances. We also know that glial cells release ions and transmitters, especially GABA (for recent references see Erdo and Wolff 1990). Who knows, the entire show of neuronal activity may be for the benefit of the glia, and not the other way around, glial cells serving the neurons! As we ponder the relationship of brain and mind, we may discover that physiologists, in their preoccupation with neurons, have looked at only part of the system.
Conclusion The search for the transmitter substance of inhibitory neurones, in which Hugh McLennan has had a major part, has led to interesting discoveries and puzzles. It led to the recognition that amino acids can qualify as transmitter substances and thus opened the way to glycine, glutamate, aspartate, taurine, and others. The implications of the fact that these amino acids are major entities in cell metabolism, as already shown by Jorge Awapara and by Eugene Roberts in the early 1950s, have not yet been fully explored. The interaction of GABA and neuropeptides, particularly where they are co-transmitters, requires far more attention than it has received so far. The exploration of the involvement of GABA in neuron -glia interactions certainly promises new insights into mode of operation of the brain. Whatever the brain does for the mind, we can be sure that GABA plays a major role in it. Acknowledgements Recent work by the author was supported by SFB 156 of the Beutsche Forschungsgemeinschafh. I wish to thank Ms, Martina Wathmayer for her excellent assistance and Ms. M. A. Cahill and Ms. G. Y. Chapman for help with the preparation of the figures and of the manuscript. A ~ s x ~ ~ ~ a o wJ.aS.c z195 , 1. Muscle receptor organs in the abdomen of Pismarus vukgaris and Palinuaus vulgaris. Q*J. Microsc. Sei. 92: 163- 199.
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