Edith Graef McGeer - Society for Neuroscience

344 Patrick L, McGeer and Edith GraefMcGeer
following neuronal systems lent themselves to mapping through enzyme
localization: the cholinergic system through choline acetyltransferase, the
GABA system through GABA transaminase, and the glutamate system
through phosphate-activated glutaminase. While developing the methodology
for GABA transaminase, we stumbled upon neurons positive for
NADPH diaphorase, later shown to be nitric oxide synthase, and these
were also mapped.
The Cholinergic System of Brain
In the days before central neurotransmitters were established, the most
promising candidate was acetylcholine. However, the definition of cholinergic
cells in brain lagged far behind that of other neurotransmitters
such as the catecholamines, serotonin, and GABA. Loewi, with his
elegantly simple 1921 experiment of setting up two frog hearts in
series, had, as Sir Henry Dale described, Vung up the curtain on
neurotransmission.' Loewi stimulated the vagus nerve of the first
heart and allowed the perfusing solution to be dripped upon the
second heart from which the vagus nerve had been cut. The second heart
slowed, and the material, first named Vagusstoff,' was identified in
1926 as acetylcholine. Quastel and associates in 1936 incubated brain
slices with glucose and oxygen in the presence of eserine. They obtained
a material indistinguishable from acetylcholine by bioassay. De Robertis
and colleagues in Argentina, and Whittaker and his team in England,
developed the technique of differential centrifugation and were able
to identify a fraction containing pinched-off nerve endings that they called
synaptosomes. They found that choline acetyltransferase, the enzyme
that synthesizes acetylcholine, occurred in the synaptosomal fraction.
Distribution studies showed a highly unequal distribution in brain,
indicating the likelihood of specific pathways using acetylcholine as the
neurotransmitter.
The method of localizing a neurotransmitter synthetic enzyme by
immunohistochemistry had been pioneered by Eugene Roberts and
colleagues. They had established GABA as a neurotransmitter by purifying
glutamic acid decarboxylase, developing antibodies to it, and using the
technique of immunohistochemistry to localize its presence in specific
pathways.
The group from our laboratory that applied this approach to the cholinergic
system was an unlikely team. Vijendra Singh, who had come from
India to take his Ph.D. under Shan Ching Sung in our laboratory, took on
the job of purifying choline acetyltransferase as his first postdoctoral
project. His efforts were crowned with the first purification of the enzyme.
He innoculated a rabbit with the protein and obtained the first antibodies
to it. By today's techniques, the antibody titer in the serum would have
been sufficiently high to do the job of brain mapping, but in the early days