On amino acid neurotransmitters in the spinal cord motor nuclei

Abstract

Using pre- and post-embedding immunocytochemistry, the distribution of six amino acid neurotransmitter candidates, with special reference to y-amino-butyric acid (GABA), glycine and glutamate was examined in boutons apposing somata and dendrites in the lumbar motor nuclei of the cat spinal cord. The monosynaptic connection between muscle spindle I a afferents and motoneurons regarding content of neuroactive amino acid and complement of amino acid receptors in the postsynaptic membrane was studied in the rat. The distribution of immunoreactivities to the amino acids GABA, glycine, glutamate, aspartate, homocysteate and taurine revealed that only GABA, glycine and glutamate were likely to be neurotransmitter candidates in the ventral horn of the cat. About 90% of all boutons apposing somata and dendrites in the L7 motor nucleus were immunoreactive to glutamate or GABA and/or glycine. There was a clear correlation between number of oold particles (amino acid immunoreactivity) overlying synaptic vesicles and the number of vesicles in immunoreactive boutons, indicating that the detected amino acid immunoreactivity represented a transmitter pool rather than a metabolic pool. In the type of aldehyde fixed tissue used here, there was a clear correlation between ultrastructural characteristics of boutons such as shape of the synaptic vesicles, and the content of amino aeid neurotransmitter. GABA and Glycin immunoreactive boutons contained mainly flat vesicles, while glutamate-immunoreactivity was seen in boutons with spherical vesicles. About 60 % of all boutons in contact with somata and dendrites in the motor nucleus contained olycine- and/or GABA immunoreactivity. Approximately 60% of these boutons were immunoreactive to glycine only, 1/3 contained both GABA and glycine, while less than 5% contained GABA-immunoreactivity only. Some 35% of the total number of boutons contained glutamate immunoreactivity. The motoneurons could with respect to the general synaptic architecture be divided in two main compartments, a proximal somajuxtasomatic compartment (including stem dendrites), and a distal dendritic compartment. This compartmentalization was relevant both with respect to composition of amino acid input and the density/size of the impinging boutons. The proximal compartment had a glycine/GABA versus glutamate ratio of 3.54, and about 10-15% of the terminals appeared not significantly labelled for any of the three amino acids examined. In the distal compartment, this ratio was only 1.5 and the fraction of not significantly labelled terminals was only half of that seen in the proximal compartment. Three weeks after intramedullary axotomy, all types of immunolabelled terminals were reduced in number, but there was a distinct shift in the ratio between the number of terminals that were immunoreactive for the inhibitory amino acids (GABA and glycine) and the number of terminals that were glutamate-immunoreactive. On the cell bodies, this ratio increased from 3.7 in normal material to 14.5 in lesioned motoneurons. As a first step to examine amino acid neurotransmitters in functionally defined circuits, enrichment of glutamate-immunoreactivity over synaptic vesicles was seen in la boutons apposing motoneurons. In la synaptic complexes with motoneurons, immunoreactivity to AMPA (GluR2/3-4) and NMDA (2A/B) receptor subunits was associated with the postsynaptic membrane specializations. These results provide further evidence for glutamate as the last transmitter in la synapses. and that released glutamate act on AMPA receptors.