Download Synaptic chemistry in single neurons: GABA is identified as an

J Neurophysiol 93: 3029 –3031, 2005;
doi:10.1152/classicessays.00026.2005.
Editorial Focus
ESSAYS ON APS CLASSIC PAPERS
Synaptic chemistry in single neurons: GABA is identified as an
inhibitory neurotransmitter
Ronald Harris-Warrick
Department of Neurobiology and Behavior, Cornell University, Ithaca, New York
TODAY WE KNOW that there are many different neurotransmitters, each interacting with its spectrum of receptors to evoke a
variety of different responses in neurons. However, in the
1950s, the idea that neurotransmission was primarily of a
chemical rather than electrical nature was still a topic for
controversy, at least in the United States. Only two neurotransmitters had been identified: acetylcholine as the transmitter of
parasympathetic and preganglionic sympathetic neurons and
spinal motoneurons, and norepinephrine as the transmitter of
sympathetic neurons. Although it was understood that there are
inhibitory as well as excitatory synapses, little was known of
what compounds, or how many of them, might mediate inhibitory transmission; in addition to its inhibitory effect on the
heart, acetylcholine appeared not to be involved. This problem
was solved for the first time by Edward Kravitz (Fig. 1) and his
colleagues in Stephen Kuffler’s group at Harvard Medical
School: in an innovative series of biochemical and physiological experiments, they proved that ␥-aminobutyric acid, or
GABA, is the inhibitory neurotransmitter at the lobster neuromuscular junction. Several of the crucial papers were published
in the Journal of Neurophysiology, and two of them are
discussed in this commentary.
GABA was identified in the brain in 1950. Its function was
unknown, but it was hypothesized to be involved in metabolism. Interest in GABA as an inhibitory neurotransmitter was
initiated by Bazemore, Elliott, and Florey, who isolated inhibitory substances from brain that could block the spontaneous
spike discharge of the crayfish stretch receptor preparation.
Starting with 52 kg of beef brain, they were able to isolate an
active extract that was called Factor I (1, 2). On further
fractionation, Florey and colleagues found that the major active
substance in Factor I was GABA, which had much higher
Address for reprint requests and other correspondence: R. Harris-Warrick, Dept. of Neurobiology and Behavior, Cornell Univ., Ithaca, NY 14853
(E-mail: [email protected]).
http://www.the-aps.org/publications/classics
specific activity than any other compound. They cautiously
concluded, “It thus seems probable that ␥-aminobutyric acid is
concerned in some aspects of the regulation of physiological
activity in the brain and other nervous structures. It is possibly
a transmitter substance of inhibitory impulses. . .” (2).
However, further work convinced Florey that he was wrong
about GABA. Attempts to purify GABA or its synthetic
enzyme from crustacean muscles or nerves were apparently
unsuccessful. Furthermore, GABA failed to mimic the strychnine-sensitive inhibitory substance in the mammalian spinal
cord (which we now know to be glycine). In an important
review in 1961, Florey (5) concluded, “There is sufficient
evidence that GABA is not the inhibitory transmitter, though it
imitates the transmitter action in many crustacean preparations.” Mammalian neurophysiologists, including David Curtis
and Hugh McClellan, had trouble mimicking the endogenous
inhibitory transmitter effects with GABA, and at a major
FIG.
1.
Edward Kravitz.
0022-3077/05 $8.00 Copyright © 2005 The American Physiological Society
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This essay looks at the historical significance of two APS classic papers that are
freely available online:
Kravitz EA, Kuffler SW, and Potter DD. Gamma-aminobutyric acid and
other blocking compounds in Crustacea. III. Their relative concentrations in
separated motor and inhibitory axons. J Neurophysiol 26: 739 –751, 1963 (http://
jn.physiology.org/cgi/reprint/26/5/739).
Otsuka M, Kravitz EA, and Potter DD. Physiological and chemical architecture of a lobster ganglion with particular reference to gamma-aminobutyrate and
glutamate. J Neurophysiol 30: 725–752, 1967 (http://jn.physiology.org/cgi/reprint/
30/4/725).
Editorial Focus
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2.
David Potter.
conference in 1959, most scientists concluded that GABA was
probably not an inhibitory transmitter (16).
A number of neurophysiologists studying the actions of
GABA at the crustacean neuromuscular junction were convinced that this conclusion was premature. These workers took
advantage of the extreme simplicity of muscle innervation in
lobster and crayfish walking legs to further study the actions of
GABA. The opener of the dactyl, for example, is only innervated by two axons, one excitatory and one inhibitory. These
can be separately stimulated, allowing a pure excitatory or a
pure inhibitory postsynaptic response to be detected and analyzed.
Kuffler and Edwards (13) and Boistel and Fatt (3) showed
that GABA mimics the action of the natural inhibitory transmitter at the crustacean stretch receptor and neuromuscular
junction, causing little change in membrane potential but a
significant decrease in input resistance. Both substances had
the same reversal potential, which was due to a selective
increase in chloride permeability. Stephen Kuffler, at Harvard,
decided to organize a group to study the neurotransmitters used
at the crustacean neuromuscular junction. Kuffler was lucky to
recruit a talented electrophysiologist, David Potter (Fig. 2), and
a talented biochemist, Edward Kravitz, who was then a postdoctoral fellow at the National Institutes of Health (NIH)
studying the metabolism of opium alkaloids. This team was
finally able to solve the GABA problem.
Four criteria had to be met before the compound could be
confirmed as a neurotransmitter. First, it had to mimic the
physiological and pharmacological properties of the natural
transmitter. This criterion had already been met for GABA at
the crustacean neuromuscular junction (3). Second, the compound had to be present in the presynaptic neuron. This was a
very tough assignment in the days before immunocytochemistry, and it was Kravitz’s job as the team biochemist to develop
the microchemical techniques to meet this criterion. First, they
showed that GABA is present in extracts of crayfish muscles
and nerves (7). In a careful screen of compounds found in the
central and the peripheral nervous system of lobsters, about ten
compounds with blocking activity at the neuromuscular junction (reducing the amplitude of evoked excitatory junctional
potentials) were isolated by water extraction, ion exchange
columns, paper chromatography, and electrophoresis (4, 9).
J Neurophysiol • VOL
GABA had by far the highest specific activity (more than 50
times higher than the next most active compound, ␤-alanine),
but it only accounted for about 20% of the total blocking
activity. Moreover, GABA was selectively concentrated in the
small bundles that only had the excitatory and inhibitory axons.
Kravitz decided that the only way to determine where
GABA was located was to separate the single excitatory and
inhibitory axons that innervate the dactyl of the walking leg
and compare the concentrations of the blocking agents in each
axon. This was a technical tour de force, described in the first
paper featured in this commentary (8). Kravitz and colleagues
carefully dissected the two axons apart from several joints of
the walking legs of several hundred lobsters. In all, 5 meters of
single axons were isolated for analysis. GABA was rapidly
isolated using ion exchange columns and electrophoresis. To
measure the tiny amounts of GABA in single axons, Kravitz
developed two new and more sensitive assays for GABA. The
first was based on the enzymatic conversion of GABA to
succinate by the enzymes GABA-glutamic acid transaminase
and succinic semialdehyde dehydrogenase, reducing NADP to
NADPH in the process, which could be measured spectrophotometrically. The enzymes were isolated from a strain of
Pseudomonas fluorescens that can grow on GABA alone as an
energy source, which Kravitz had carried in his pocket to
Harvard from the NIH. When there was insufficient GABA for
this assay (1 nmol), a more sensitive fluorometric assay was
developed where the newly synthesized NADPH was isolated
and reconverted back to NADP for measurement; this method
was sensitive down to 10 pmol. The very first experiments
were startling: significant amounts of GABA were found in the
inhibitory axons, whereas none was detectable from the excitatory axons. Further experiments showed that there is indeed
a small amount of GABA (with still unknown function) in the
excitatory motoneurons, but the ratio of GABA in the inhibitory to excitatory axons was about 100:1, with about 0.1 M
GABA in the inhibitory axons (11).
In another tour de force of single cell biochemistry, described in the second paper in this commentary (15), Otsuka,
Kravitz, and Potter proceeded to compare levels of GABA in
single identified neurons in lobster abdominal ganglia. Again,
the lobster proved to be an ideal choice for these studies. The
abdominal ganglion contains a number of very large neurons in
stereotyped positions; these neurons do not have any synapses
on their somata, so the isolated soma was not contaminated by
transmitters in adherent presynaptic terminals. Neurophysiological studies were carried out to identify 21 pairs of neurons
that innervate peripheral neuromusclar junctions: three pairs
were inhibitory, innervating different muscles, whereas the rest
were excitatory motoneurons. Kravitz and his colleagues developed a novel method to isolate these single neurons and
assay their levels of GABA using the sensitive fluorometric
assay. Almost none of the excitatory motoneurons had any
detectable GABA by this assay, whereas all of the inhibitory
neurons tested contained GABA. This was the first study of the
chemical architecture of a region of the nervous system and
stands as a model for studies that followed.
Although these papers unambiguously showed that GABA is
highly concentrated in inhibitory neurons compared with excitatory neurons, Kravitz and colleagues were not satisfied that
they had proven that GABA is the inhibitory neurotransmitter
of these neurons. Two more criteria for identification of a
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FIG.
ESSAYS ON APS CLASSIC PAPERS
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ESSAYS ON APS CLASSIC PAPERS
J Neurophysiol • VOL
elsewhere. Invertebrates have a long and successful record for
major discoveries in neuroscience (for example, the ionic basis
of the action potential, organization of small motor networks,
mechanisms of neuromodulation, and the cellular basis of
learning and memory), and they will continue to serve this
function into the future.
REFERENCES
1. Bazemore A, Elliott KAC, and Florey E. Factor I and ␥-aminobutyric
acid. Nature 178: 1052–1053, 1956.
2. Bazemore AW, Elliott KAC, and Florey E. Isolation of Factor I.
J Neurochem 1: 334 –339, 1957.
3. Boistel J and Fatt P. Membrane permeability change during inhibitory
transmitter action in crustacean muscle. J Physiol 144: 176 –191, 1958.
4. Dudel J, Gryder R, Kaji A, Kuffler SW, and Potter DD. Gammaaminobutyric acid and other blocking compounds in Crustacea. I. Central
nervous system. J Neurophysiol 26: 721–728, 1963.
5. Florey E. Comparative physiology: transmitter substances. Annu Rev
Physiol 23: 501–528, 1961.
6. Hall ZW, Bownds MD, and Kravitz EA. The metabolism of gamma
aminobutyric acid in the lobster nervous system. J Cell Biol 46: 290 –299,
1970.
7. Kravitz EA. Enzymic formation of gamma-aminobutyric acid in the
peripheral and central nervous system of lobsters. J Neurochem 9: 363–
370, 1962.
8. Kravitz EA, Kuffler SW, and Potter DD. Gamma-aminobutyric acid and
other blocking compounds in Crustacea. III. Their relative concentrations
in separated motor and inhibitory axons. J Neurophysiol 26: 739 –751,
1963.
9. Kravitz EA, Kuffler SW, Potter DD, and Van Gelder NM. Gammaaminobutyric acid and other blocking compounds in Crustacea. II. Peripheral nervous system. J Neurophysiol 26: 729 –738, 1963.
10. Kravitz EA, Molinoff PB, and Hall ZW. A comparison of the enzymes
and substrates of gamma-aminobutyric acid metabolism in lobster excitatory and inhibitory axons. Proc Natl Acad Sci USA 54: 778 –782, 1965.
11. Kravitz EA and Potter DD. A further study of the distribution of
␥-aminobutyric acid between excitatory and inhibitory axons of the
lobster. J Neurochem 12: 323–328, 1965.
12. Kravitz EA, Potter DD, and Van Gelder NM. Gamma-aminobutyric
acid and other blocking substances extracted from crab muscle. Nature
194: 382–383, 1962.
13. Kuffler S and Edwards C. Mechanism of gamma-aminobutyric acid
(GABA) action and its relation to synaptic inhibition. J Neurophysiol 21:
589 – 610, 1958.
14. Otsuka M, Iversen LL, Hall ZW, and Kravitz EA. Release of gammaaminobutyric acid from inhibitory nerves of lobster. Proc Natl Acad Sci
USA 56: 1110 –1115, 1966.
15. Otsuka M, Kravitz EA, and Potter DD. Physiological and chemical
architecture of a lobster ganglion with particular reference to gammaaminobutyrate and glutamate. J Neurophysiol 30: 725–752, 1967.
16. Roberts E and Baxter DF. Inhibition in the Nervous System and GammaAminobutyric Acid. New York: Pergamon, 1960.
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transmitter remained to be met. The third criterion was to show
that the transmitter is synthesized in the presynaptic neuron.
This criterion was met by demonstrating the enzymatic synthesis of GABA from radioactive glutamate by glutamic acid
decarboxylase (GAD), first in whole central or peripheral
nervous tissue (7) and then in single excitatory and inhibitory
axons (6, 7). These experiments demonstrated that the GAD
concentration in inhibitory axons is at least 100 times higher
than in the excitatory axons. The metabolic enzymes that break
down GABA were found in similar amounts in the two nerves.
The fourth and final criterion was to show that GABA is
released from the inhibitory nerve terminal on stimulation.
Again taking advantage of the simplicity of the lobster neuromuscular junction, Otsuka et al. (14) superfused opener muscles in the large crusher claw of lobsters while stimulating the
single excitatory and inhibitory nerves innervating the muscle.
GABA was isolated from the saline superfusing the muscle
using ion exchange columns and was measured using the
enzyme assay. On stimulation of the inhibitory axon, significant amounts of GABA were released, and the amount rose
with the number of stimuli. Release was calcium dependent, as
expected for a neurotransmitter. In contrast, no detectable
GABA was released when the excitatory motoneuron was
selectively stimulated. In further work, Kravitz and his students
characterized all the enzymes in the GABA synthetic and
metabolic pathway and finally showed that there is a specific
reuptake mechanism for GABA that could serve to inactivate
the released GABA. Throughout this work, Kuffler, Kravitz,
and their colleagues were very careful not to overstate their
case. It was only after all of these results were obtained that
Kravitz finally wrote, without any qualifications, “␥-aminobutyric acid (GABA) is the inhibitory transmitter compound at
the lobster neuromuscular junction.” (6).
These experiments proved conclusively for the first time that
GABA is an inhibitory neurotransmitter. As the results obtained by Kravitz and his very able colleagues (Otsuka,
Iversen, Hall, Potter) supporting this accumulated, they were
for the most part ignored by vertebrate neuroscientists. It was
only a decade later that similar experiments were possible in
the vertebrate central nervous system, verifying that GABA is
indeed the most common inhibitory neurotransmitter in the
brain, although certainly not the only one. Taking advantage of
the numerical simplicity of the invertebrate nervous system,
Kravitz and his collaborators were able to demonstrate a
general principle of neuronal function that could not be proven
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