Abstract
Motoneuron synapses on spinal cord interneurons known as Renshaw cells activate nicotinic, AMPA and NMDA receptors consistent with co-release of acetylcholine and excitatory amino acids (EAA). However, whether these synapses express vesicular glutamate transporters (VGLUTs) capable of accumulating glutamate into synaptic vesicles is controversial. An alternative possibility is that these synapses release other EAAs, like aspartate, not dependent on VGLUTs. To clarify the exact EAA concentrated at motor axon synapses we performed a quantitative postembedding colloidal gold immunoelectron analysis for aspartate and glutamate on motor axon synapses (identified by immunoreactivity to the vesicular acetylcholine transporter; VAChT) contacting calbindin-immunoreactive (-IR) Renshaw cell dendrites. The results show that 71% to 80% of motor axon synaptic boutons on Renshaw cells contained aspartate immunolabeling two standard deviations above average neuropil labeling. Moreover, VAChT-IR synapses on Renshaw cells contained, on average, aspartate immunolabeling at 2.5 to 2.8 times above the average neuropil level. In contrast, glutamate enrichment was lower; 21% to 44% of VAChT-IR synapses showed glutamate-IR two standard deviations above average neuropil labeling and average glutamate immunogold density was 1.7 to 2.0 times the neuropil level. The results were not influenced by antibody affinities because glutamate antibodies detected glutamate-enriched brain homogenates more efficiently than aspartate antibodies detecting aspartate-enriched brain homogenates. Furthermore, synaptic boutons with ultrastructural features of Type I excitatory synapses were always labeled by glutamate antibodies at higher density than motor axon synapses. We conclude that motor axon synapses co-express aspartate and glutamate, but aspartate is concentrated at higher levels than glutamate.
Highlights
The release of acetylcholine from motor axons at the mammalian neuromuscular junction (NMJ) has been established for more than 75 years [1], but recent studies suggest that additional neurotransmitters, in particular excitatory amino acids (EAAs) like glutamate, might be co-released from motoneuron synapses both in the periphery and centrally
The major finding in this study is that motor axon synapses on Renshaw cells are enriched in both aspartate and glutamate, but at different levels
Mechanisms for the accumulation of glutamate and aspartate inside synaptic vesicles The fact that aspartate and glutamate immunoreactivities are enriched in areas within synaptic boutons containing high density of synaptic vesicles, including presynaptic active zones, suggests that these amino acids could be accumulated in a releasable pool
Summary
The release of acetylcholine from motor axons at the mammalian neuromuscular junction (NMJ) has been established for more than 75 years [1], but recent studies suggest that additional neurotransmitters, in particular excitatory amino acids (EAAs) like glutamate, might be co-released from motoneuron synapses both in the periphery and centrally. In vitro studies (spinal cord slices or whole neonatal spinal cords) failed to fully inhibit Renshaw cell-mediated disynaptic recurrent inhibition of motoneurons or motor axon excitatory postsynaptic currents (EPSCs) on Renshaw cells with acetylcholine [13,14]. In this case receptor antagonists were bath applied to either isolated spinal cords or spinal cord slices, an experimental situation believed to result in better saturation of postsynaptic receptors by antagonists. Late Renshaw cell discharges in response to motor axon input were shown to be NMDA-dependent in the neonatal spinal cord [15]
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