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Journal of Neuroscience, Vol 11, 2087-2101, Copyright © 1991 by Society for Neuroscience
Glutamate and aspartate immunoreactivity in hypothalamic presynaptic axons
AN van den Pol
Section of Neurosurgery, Yale University School of Medicine, New Haven, Connecticut 06510.
Within the hypothalamus, a large number of neuroactive substances are
found, many first detected in this part of the brain. Excitatory amino
acids, recognized as important transmitters in other parts of the brain,
have received little attention here. To study glutamate immunoreactivity at
the ultrastructural level in the hypothalamus, postembedding colloidal gold
or silver-intensified gold was used. Antisera raised against glutamate
conjugated with glutaraldehyde to keyhole limpet hemocyanin were specific
for glutamate, tested with a battery of tests including immunodot blot,
ELISA assays. Western blot, and Sepharose epoxy-conjugated amino acids.
Antisera did not cross- react with other amino acids and related compounds,
with proteins containing glutamate, or with polyglutamate. A population of
presynaptic boutons in the suprachiasmatic, arcuate, ventromedial,
supraoptic, and parvocellular and magnocellular paraventricular nuclei
showed strong immunoreactivity for glutamate. Highly labeled presynaptic
axons generally made asymmetrical Gray type 1 synaptic contacts with
dendrites or cell bodies and had up to eight times more immunogold
particles per unit area than postsynaptic dendrites. Axon terminals
exhibiting strong glutamate immunoreactivity had large numbers of round,
clear vesicles adjacent to the synaptic specialization together with a few
larger, dense-core vesicles. The largest number of gold particles over
axons were located in regions containing the small clear vesicles. Axons in
general had about three times more gold particles over them than did the
postsynaptic dendrites. Staining of single boutons in adjacent serial
ultrathin sections with glutamate or GABA antisera showed that
non-GABAergic terminals had a higher level of glutamate staining than did
axons immunoreactive for GABA. In control experiments, immunostaining with
glutamate antiserum could be blocked by solid-phase absorption of the
antiserum with glutamate conjugated with glutaraldehyde to proteins.
Aspartate was also detected with immunocytochemistry in some presynaptic
boutons in the medial hypothalamus. To compare the response of neurons to
aspartate and glutamate, calcium-imaging dyes were used in combination with
digital video microscopy. Whereas almost all neurons showed a rise in
intracellular Ca2+ in response to glutamate, many but not all of the same
cells also showed a Ca2+ rise of smaller magnitude in response to
aspartate. These ultrastructural immunocytochemical data, taken in
conjunction with biochemical and electrophysiological experiments, suggest
that glutamate, and to a lesser extent aspartate, may play an important
neurotransmitter role in a wide variety of hypothalamic circuits.
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