Qualitative and quantitative analysis of glycine- and GABA-immunoreactive nerve terminals on motoneuron cell bodies in the cat spinal cord: A postembedding electron microscopic study

Abstract

The distribution of glycine- and gamma-aminobutyric acid (GABA)-like immunoreactivity (LI) in nerve terminals on the cell soma of motoneurons in the aldehyde-fixed cat L7 spinal cord was examined using postembedding immunogold histochemistry in serial ultrathin sections. Quantitative examination of 405 terminals on eight neurons of alpha-motoneuron size in the L7 motor nuclei from one animal was performed. A majority of the terminals (69%) were immunoreactive to glycine and/or GABA. These terminals contained flat or oval synaptic vesicles, thus classifying them as F type or as C type in one case. In no case was a type-F terminal unlabeled for both glycine and GABA. Most of the immunolabeled terminals were immunoreactive to glycine only (62.5%), whereas 35.4% contained both glycine- and GABA-LI. A very small number of immunolabeled terminals (2%) were immunoreactive to GABA only. In those terminals, where glycine- and GABA-LI coexisted, the gold particle density for each amino acid was only half of that seen in boutons containing only one of the two amino acids. The involvement of glycine and GABA in postsynaptic inhibition of spinal alpha-motoneurons is discussed, with particular reference to the possibility that these two inhibitory amino acids may be coreleased from a significant proportion of the nerve terminals impinging on the cell bodies.

P boutons in lamina IX of the rodent spinal cord express high levels of glutamic acid decarboxylase-65 and originate from cells in deep medial dorsal horn

“…Most of the latter express the glycine transporter GLYT2 and therefore presumably use both GABA and glycine as transmitters, whereas the GAD65-intense boutons lack GLYT2 (6). Consistent with this observation, it has been reported that P boutons in lamina IX are GABA-immunoreactive, but not glycine-immunoreactive, whereas many other GABAergic boutons in this lamina are enriched with glycine (10,22). The presence of clusters of GAD65-intense boutons in all motor nuclei in the L3-L5 segments implies that Ia afferent input from all hindlimb muscles is under presynaptic control by P boutons.…”

“…He also demonstrated that the postsynaptic elements were of primary afferent origin because they degenerated after dorsal rhizotomy (20). It was subsequently shown that P boutons are presynaptic to terminals of Ia afferents (7)(8)(9)(10) and that they are immunoreactive with Abs against GAD (2) and GABA (10,16,21,22). These findings are consistent with the suggestion that GABAergic axo-axonic synapses are the anatomical basis for the presynaptic inhibition of Ia afferents and the accompanying primary afferent depolarization (23).…”

“…There are two lines of evidence to suggest that all P boutons belong to the GAD65-intense population. Because P boutons are strongly GABA-immunoreactive (10,16,22), they must contain at least one GAD isoform. Although we saw many axons with significant levels of GAD67 in lamina IX, these were very seldom adjacent to VGLUT1-labeled boutons, which include all proprioceptive afferents (11).…”

“…This observation is compatible with a triadic arrangement involving P boutons because these are GABAergic but not glycinergic. Despite their role in producing postsynaptic inhibition of motoneurons at synaptic triads, the P boutons clearly form a different functional population from the majority of GABAergic boutons in the ventral horn, which form axo-somatic and axodendritic synapses, and which include some of the F boutons that are presynaptic to motoneurons (18,21,22). Recent studies have identified four classes of ventral interneurons in the developing neural tube (26).…”

GABA‐, glycine‐, and glutamate‐immunoreactive bouton profiles in apposition to neurons of the central cervical nucleus in the rat

“…One section was kept to examine the B-HRP reaction product, and adjacent sections were incubated with one of the following three primary antisera, all raised in rabbit: anti-GABA (1:600), anti-glycine (1:300), or anti-glutamate (1:400; Glu #03), using previously described postembedding immunocytochemical procedures (Craig, 1971;Storm-Mathisen et al, 1983;van Lookeren Campagne et al, 1991;Davanger et al, 1994). These antisera were characterized previously (Storm-Mathisen et al, 1983;Shupliakov et al, 1993;Davanger et al, 1994;Ö rnung et al, 1994;Ö rnung et al, 1996). As an additional specificity control, a test system of plastic-embedded amino acid conjugates was incubated along with the tissue sections (Ottersen, 1987;Zhang et al, 1992;Ö rnung et al, 1996).…”