VESICLE SHAPE AND AMINO-ACIDS IN SYNAPTIC INPUTS TO PHRENIC MOTONEURONS - DO ALL INPUTS CONTAIN EITHER GLUTAMATE OR GABA

Varicosities that made synapses or direct contacts with retrogradely l abelled rat phrenic motoneurons were examined for their content of imm unoreactivity for either glutamate or glutamate decarboxylase, the enz yme involved in synthesis of gamma-aminobutyric acid (GABA). Phrenic m otoneurons were identified by retrograde tracing from the diaphragm wi th cholera toxin B subunit conjugated to horseradish peroxidase. Cell bodies and medium-sized to large dendrites were labelled. Preembedding immunocytochemistry identified glutamate decarboxylase-immunoreactive nerve fibres; glutamate-immunoreactive nerve terminals were identifie d using postembedding immunogold labelling of ultrathin sections. The presence of glutamate- or glutamate decarboxylase immunoreactivity in nerve terminals was correlated with the morphology of the synaptic ves icles. Two major classes of nerve terminals were identified. Nerve ter minals with round (presumably spherical) synaptic vesicles (S terminal s) comprised 55% of synapses and contacts on phrenic motoneuron somata and 58% of synapses and direct contacts with dendrites. Nerve termina ls with flattened synaptic vesicles (F terminals) comprised 42% of syn apses direct contacts with somata and 41% of synapses and direct conta cts with dendrites. Analysis of immunogold-labelled sections showed th at S terminals contained statistically higher levels of glutamate immu noreactivity than F terminals. At the light microscope level, many glu tamate decarboxylase-immunoreactive nerve terminals surrounded retrogr adely labelled motoneurons. Varicosities with glutamate decarboxylase immunoreactivity made 33% of all synapses and direct contacts on somat a, and 33% of synapses and direct contacts with dendrites of the retro gradely labelled phrenic motoneurons. Flattened synaptic vesicles were present in those glutamate decarboxylase-immunoreactive nerve termina ls in which synaptic vesicle morphology could be judged. An additional 10% of all nerve terminals were of the F type, but were not glutamate decarboxylase-immunoreactive. Three percent of terminals on somata an d 1% of nerve terminals on dendrites could not be classified as S or F types. These findings suggest that more than 90% of all inputs to phr enic motoneuron cell bodies and proximal dendrites could contain eithe r GABA or glutamate. Some of these glutamatergic and GABAergic nerve f ibres undoubtedly represent the source of inspiratory drive to, or exp iratory inhibition of, phrenic motoneurons. (C) 1996 Wiley-Liss, Inc.