Journal of Neuroscience, Vol 12, 3789-3803, Copyright © 1992 by Society for Neuroscience
Immunogold quantification of glutamate in two types of excitatory synapse with different firing patterns
O Shupliakov, L Brodin, S Cullheim, OP Ottersen and J Storm-Mathisen
Nobel Institute for Neurophysiology, Karolinska Institutet, Stockholm, Sweden.
A quantitative immunocytochemical method was used to study the regional
levels of glutamate in two types of lamprey (Ichtyomyzon unicuspis) axon,
which both activate excitatory amino acid receptors, but which when active
exhibit different firing patterns. Giant reticulospinal axons fire in brief
bursts, while dorsal column axons, mainly belonging to cutaneous afferents,
show a sustained firing at high frequency. In both types of axon, clusters
of synaptic vesicles showed a strong accumulation of glutamate immunogold
labeling, and the density of gold particles correlated strictly with the
packing density of synaptic vesicles. The most densely packed vesicle areas
had a particle density corresponding to a concentration of fixed glutamate
of about 30 mM in coprocessed glutamate conjugates, suggesting an
intravesicular glutamate concentration of at least 60 mM. The level of
labeling in axoplasmic matrix was considerably lower, but differed
significantly between the two types of axon. Dorsal column axons showed a
particle density in axoplasmic matrix that was approximately four times
higher than that in giant reticulospinal axons. The mitochondrial glutamate
labeling was also significantly stronger in the dorsal column axons. In
addition, the number of mitochondrial profiles surrounding vesicle clusters
was about four times higher in dorsal column synapses than in
reticulospinal synapses. Antisera to aspartate, GABA, glutamine, and
homocysteate failed to produce a specific labeling of synaptic vesicle
clusters in reticulospinal or dorsal column axons. In conjunction with
previous demonstrations of a stimulus-induced vesicle depletion in giant
reticulospinal synapses (Wickelgren et al., 1985), these results imply that
glutamate is released from synaptic vesicles. The different extravesicular
glutamate levels in reticulospinal axons and dorsal column axons may relate
to different requirements for the refilling of synaptic vesicles in these
functionally divergent neurons.
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