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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.

The present study was conducted to examine the plasticity of 5-hydroxytryptamine (5-HT)-immunoreactive terminals in the rat phrenic nucleus following an ipsilateral C2 spinal cord hemisection and 30-day survival period. A retrograde horseradish peroxidase (HRP) labeling technique was used to identify the phrenic motoneurons at the electron microscopic (EM) level. After employing a pre-embedding immunocytochemical technique, the ultrastructural characteristics of 5-HT-immunoreactive terminals were qualitatively and then quantitatively analyzed with a computerized morphometric system before and after injury in separate groups of rats. The results indicated that the majority of the 5-HT-labeled terminals formed axodendritic contacts, but some 5-HT-labeled terminals made axosomatic contacts. 5-HT terminals were associated with either asymmetrical or symmetrical synapses, and some displayed postsynaptic dense bodies. Approximately 2% of the 5-HT terminals had dense-core vesicles. Although the total number of labeled and unlabeled terminals in the phrenic nucleus was reduced after hemisection, the number of 5-HT terminals in the hemisected group was greater than that of the control group. Moreover, the total number and length of asymmetrical and symmetrical synaptic active zones per 5-HT terminal were significantly greater after injury. Finally, the total number of 5-HT terminals with multiple synapses was significantly greater in the hemisected group as compared to controls. It is possible that 5-HT synaptic plasticity may be part of the morphological substrate for the unmasking of the latent crossed phrenic pathway which mediates recovery of the ipsilateral hemidiaphragm paralyzed by C2 spinal cord hemisection.

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The present study was designed to: 1) morpholog ically characterize cat glutamate and GABAergic synaptic term inals in lamina IX in the intact spinal cord at the electron microscopic leve l using postembedding immunochemical techniques and 2), begin an analysis of how the synaptic architecture of glutamate and GABAergic termi nals changes after an ipsilateral spinal cord hemisection. The present study shows that glutamate immunoreactive terminals are characte rized by a wide synaptic cleft, asymmetric synaptic membrane densities and spherical synaptic vesicles. Most of the glutamatergic terminals are presynaptic to small or medium size dendrites. In contrast, GABAergic terminals display typical pleomorphic ·synaptic vesicles , a narrow synaptic cleft and a symmetrical membrane density. Qualitative ana lys is indicated that 13-17 months after hemisection , the length of the s ynaptic active zones in both glutamatergic a nd GABAergic terminals ipsilateral to hemisection is longer tha n those observed in the terminals cont ra lateral to hemisection or in normal control cats. Furthermore, the perimeters of both dendrites and either glutamate or GABA immunoreactive terminals are longer on the hemisected side compared with those observed in the nonhemisected s ide of the spina l co rd . These results are important for complete understand ing of the mechanisms which unde rlie locomotor recovery in mammals following spinal cord injury. (J Spinal Cord Med 1997;20:311 -318)