Ultrastructural quantitative analysis of glutamatergic and GABAergic synaptic terminals in the phrenic nucleus after...

Quantitative analysis of electron microscopic postembedding immunochemically stained material indicates that 48% of all terminals in the rat phrenic nucleus are glutamatergic and 33% are gamma-aminobutyric acid (GABA)ergic. Three distinct types of glutamatergic terminals were observed in the rat phrenic nucleus: terminals characterized by large, loosely arranged spherical synaptic vesicles (SI) or small, compact spherical synaptic vesicles (Ss) and elongated terminals containing spherical synaptic vesicles with neurofilaments (NFs). All three types of glutamatergic terminals display asymmetrical synaptic membrane densities with postsynaptic dense bodies being present in some of the S-type terminals. The GABAergic immunoreactive terminals in the phrenic nucleus most closely resemble F-type terminals. They are characterized by flattened or pleomorphic synaptic vesicles and symmetric synaptic membrane densities. Among the 48% glutamatergic terminals, 27% are SI, 65% are Ss, and 8% are NFs, respectively. Significantly fewer glutamate, GABA, and unlabeled terminals per unit area are present in the phrenic nucleus 30 days after a C2 spinal cord hemisection as compared to nonhemisected controls. The average number of active zones per terminal, however, is greater in the hemisection group (1.45 +/- 0.03) than in the control group (1.34 +/- 0.03), with the active zones in the glutamate terminals mainly accounting for this difference. Moreover, the length of the active zones in the glutamate terminals was significantly longer in the hemisection group (0.37 +/- 0.013 microns) as compared to the controls (0.24 +/- 0.008 microns). In addition, the mean length of synaptic active zones in GABAergic terminals was also found to be longer in the hemisection group (0.36 +/- 0.022 microns) as compared to controls (0.28 +/- 0.014 microns). Finally, there is also a significantly higher ratio of synaptic active zones to the total number of glutamate-labeled terminals after injury (1.73 +/- 0.08) as compared to controls (1.41 +/- 0.04). The number of double/multiple synapses, the percentages of Sl, Ss, and NFs-type terminals, and the percentages of synaptic active zones contacting either distal dendrites or proximal dendrites/somata do not change significantly 30 days after injury. These results are important for a more complete understanding of the synaptic plasticity that occurs in the phrenic nucleus after spinal cord injury and to show how the plasticity may relate to the unmasking of latent bulbospinal respiratory connections which restore function to the hemidiaphragm paralyzed by an ipsilateral spinal cord hemisection.