Identification and characterization of rat orbicularis oculi motoneurons using confocal laser scanning microscopy

The eyeblink reflex is one of the most extensively studied behaviors in mammals. The active downward force that causes lid closure is controlled by the orbicularis oculi (OO) muscle. To augment our studies on the neurophysiology and plasticity of the rat eyeblink circuit, here we present the first anatomical paper to focus exclusively on identifying and characterizing the OO motoneurons of the rat facial motor nucleus (FMN). One thousand and twenty-nine cells from four animals were retrogradely labeled by injecting the OO muscle with HRP and were imaged conventionally. One hundred and one cells from five animals were labeled by injecting the OO muscle with a 3000 mol. wt. fluorescent dextran and were imaged using confocal laser scanning microscopy (CLSM). The latter method resulted in little tissue shrinkage, bright labeling, and excellent resolution of the soma, dendrites, and axon. Furthermore, it is a histologically simple alternative to HRP for retrograde labeling from the neuromuscular junction. Both methods revealed that the OO motoneurons were distributed over the entire length of the FMN, that they were concentrated along the dorsal crest of the nucleus, and that they were less numerous in the extreme rostral and caudal regions. As measured using the CLSM method, cell body areas were highly variable, ranging from 317 to 1500 microm2, but there was no size gradient along the rostrocaudal extent of the FMN. The neurons exhibited seven primary dendrites on average, which gave rise to bifurcating and even trifurcating secondary dendrites. Using the HRP method, the estimated area of OO motoneurons ranged from 161 to 1381 microm2. The combined methods furnished a detailed characterization of the number, spatial distribution, and morphology of rat OO motoneurons. Moreover, these methods provide a useful way to analyze the circuitry that modulates the rat eyeblink.