Morphological and immunohistochemical characterization of interneurons within the rat trigeminal motor nucleus

Three series of experiments were carried out to characterize interneurons located within the trigeminal motor nucleus of young rats aged 5-24 days. Cholera toxin injections were made bilaterally into the masseter and, sometimes, digastric muscles to label motoneurons. In the first set of experiments, thick slices were taken from the pontine brainstem and cholera toxin-positive and cholera toxin-negative neurons located inside the trigeminal motor nucleus were filled with biocytin through whole-cell recording patch electrodes. Positively identified motoneurons (cholera toxin+) of various shapes and sizes always had a thick, unbranched axon that entered the motor root following a tight zigzag course. Many cholera toxin-negative neurons were also classified as motoneurons after biocytin filling based on this particularity of their axon. These are probably either fusimotor motoneurons or motoneurons supplying other jaw muscles. The cholera toxin-negative neurons classified as interneurons differed markedly from motoneurons in that they had thin, usually branched axons that supplied the ipsilateral reticular region surrounding the trigeminal motor nucleus (peritrigeminal area), the main trigeminal sensory nucleus, the trigeminal mesencephalic nucleus, the medial reticular formation of both sides, and the contralateral medial peritrigeminal area. Most often, their dendrites were arranged in bipolar arbors that extended beyond the borders of the trigeminal motor nucleus into the peritrigeminal area. Immunohistochemistry against glutamate, GABA and glycine was used to further document the nature and distribution of putative interneurons. Immunoreactive neurons were uniformly distributed throughout the rostro-caudal extent of the trigeminal motor nucleus. Their concentration seemed greater toward the edges of the nucleus and they were scarce in the digastric motoneuron pool. Glutamate- outnumbered GABA- and glycine-immunoreactive neurons. There was no clear segregation between the three populations. In the final experiment, 1,1′-dioctadecyl-3,3,3′,3′-tetra-methylindocarbocyanine perchlorate crystals were inserted into one trigeminal motor nucleus in thick slices and allowed to diffuse for several weeks. This procedure marked commissural fibers and interneurons in the contralateral trigeminal motor nucleus. Together these results conclusively support the existence of interneurons in the trigeminal motor nucleus.