The functional anatomy and evolution of hypoglossal afferents in the leopard frog, Ranapipiens

Abstract

Previously, we suggested that afferents are present in the hypoglossal nerve of the leopard frog, Ranapipiens. The basis for this was behavioral data obtained after transection of the hypoglossal nerve. These afferents coordinate the timing of tongue protraction with mouth opening during feeding. The goal of the present study was to define anatomically these hypoglossal afferents in Ranapipiens. Retrograde tracing was performed using horseradish peroxidase, fluorescent dextran amines and neurobiotin. Data show that the cell bodies of hypoglossal afferents are located in the dorsal root ganglion of the third spinal nerve and enter the brainstem through its dorsal root. The afferents ascend in the dorsomedial funiculus and move laterally after they pass the obex. They project in the granular layer of the cerebellum and the medial reticular formation. The cervical afferents that travel in this pathway are known to carry proprioceptive and cutaneous sensory information. We hypothesize that the presence of afferents in the hypoglossal nerve is a derived characteristic of anurans, which has resulted from the re-routing of afferent fibers from the third spinal nerve into the hypoglossal nerve. The appearance of hypoglossal afferents coincides with the morphological acquisition of a highly protrusible tongue.

Introduction

During evolutionary changes in morphology, neuronal pathways may be reconfigured to control the new structures, and novel sensory mechanisms may be required to coordinate their movements. Among anuran amphibians, the acquisition of a long, protrusible tongue represents such a phylogenetic event. The primitive condition for frogs is a tongue that protrudes only slightly beyond the tips of the mandibles [20]. A highly protrusible tongue has evolved several times independently [22]. Therefore, an interesting system has emerged to investigate evolutionary changes in the neural control of the highly protrusible tongues. In this study, we address the phylogenetic origins and anatomical projections of sensory neurons that coordinate tongue movements in the leopard frog, Ranapipiens.

The tongues of frogs, in all but a few cases, are attached anteriorly, and are protracted and retracted by the contraction of the extrinsic muscles, mm. genioglossus and hyoglossus. The m. genioglossus rotates the tongue over the mandibular symphysis and out of the mouth 11, 12, 22and the m. hyoglossus pulls the tongue back into the mouth. The protractor and retractor muscles of the tongue are controlled by motoneurons of the hypoglossal nerve.

The hypoglossal (CN XII) motoneurons of vertebrates have been well documented 4, 6, 17, 25, 31. Rami of the hypoglossal nerve innervate the neck musculature and the lateral muscles of the otic capsule. Ultimately, the terminal branches (ramus hypoglossus) innervate the hypobranchial muscles, including the hyoglossal and genioglossal muscles 5, 13. In extant amphibians, the brainstem has become compressed and, with the exception of a spino-occipital nerve present in a few urodele amphibians and caecilians which is composed in part by the hypoglossal nerve, the hypoglossal is formed from the combined branches of the first and second spinal nerves 3, 6.

Urodele amphibians represent the primitive condition for tetrapods; the hypoglossal nerve is a purely motor nerve and no afferent fibers are associated with it [27]. However, hypoglossal afferents have evolved several times independently among vertebrates. Because the hypoglossal nerve does not have a dorsal root ganglion, several investigators have demonstrated alternative pathways by which anastomoses from other nerves may contribute afferent fibers to the ramus hypoglossus (for review, see [17]). In primates, the hypoglossal afferents are re-directed cervical spinal neurons as well as neurons from the nodose ganglion of the vagus nerve and are used in modulation of tongue movements during vocalization [7]. These neurons enter the tongue through the hypoglossal nerve. In the dog and cat, Zimney et al. [36]found a small number of afferent fibers in the proximal vagus nerve that show degeneration following transection of the hypoglossal nerve. Among finches, Wild [35]has shown that Herbst corpuscles and terminal cell receptors of the tongue papillae are innervated by the lingual branch of the hypoglossal nerve. Their cell bodies are located in the jugular ganglion and then ascend within the trigeminal tract.

In the leopard frog, Ranapipiens (Family Ranidae), Stuesse et al. [32]showed that the distal portion of the hypoglossal nerve contains afferents, although neither the root in which the fibers entered the brainstem, nor their projections were described.

Previous studies of feeding motor patterns in Ranapipiens have shown that hypoglossal afferents coordinate the timing of tongue movements [1]. When feeding on small sizes of prey, the hypoglossal nerve must be intact for the mouth to open during feeding. When the hypoglossal nerve is transected, the frogs lunge forward and attempt to capture the prey, but the mouth does not open during the feeding cycle. Hypoglossal afferents have also been shown to gate incoming visual information and influence the choice of motor programs used during prey capture [2]. Therefore, sensory information traveling within the hypoglossal nerve presumably carries information about movements of the tongue and is used to coordinate tongue protraction and mouth opening [2].

In more basal lineages of frogs, the hypoglossal nerve appears to be purely motor. Transection of the hypoglossal nerve does not prevent these frogs from opening their mouths during feeding 8, 23. Therefore, we hypothesize that the presence of afferents in the hypoglossal nerve represents a derived condition among anurans. The appearance of these afferents coincides with the morphological acquisition of a highly protrusible tongue. In addition, a phylogenetic survey of hypoglossal afferents among anurans suggests that these afferents have evolved multiple times independently in the ranids and the bufonids [24].

In the present study, we trace hypoglossal afferent fibers from the tongue into the brainstem and illustrate the ascending projections to the cerebellum and reticular formation. We discuss the evolution of afferents from the third spinal nerve which have become re-routed to anastomose with the hypoglossal nerve. Their function is to control the timing of tongue and jaw movements in the frog, Ranapipiens.