Labeling afferent nerves in the tongue by peripheral transganglionic transport of HRP and WGA-HRP in the rat

Peripheral transganglionic transport of horseradish pcroxidase(HRP) and wheat germ agglutinin–horseradish peroxidaseconjugate (WGA–HRP) was used to label afferent fibersin the taste buds and lingual epithelium of the rat. Microinjectionsof the tracer were made in the brain stem central projectionarea of the afferent nerves to the tongue. Optimal labelingof nerve endings in the tongue was obtained when 2 µlof 20% HRP was injected into the brain stem and postinjectionsurvival times of 24–36 h were used. The distributionof single nerves was studied by using this tracing procedurein combination with strategic transections of the various afferentnerves supplying the tongue. Labeled nerve fibers from the combinedchorda tympani–lingual nerve were found in the epitheliumand in taste buds in the fungiform and anterior foliate papillaeof the anterior 3/4 of the tongue. Labeled nerve fibers in theepithelium of the anterior 2/3 of the tongue but none in tastebuds were found when the lingual nerve alone was studied, althoughnumerous perigeminal fibers were found. The glossopharyngealnerve was found to innervate die posterior 1/4 of the tongueepithelium including the taste buds of the circumvallate papillae.The glossopharyngeal nerve on one side was found to innervatethe taste buds on both sides of the midline. The results showthat this tracing procedure can be a useful supplement to othermethods for studying afferent nerves in the tongue.     

The origin of slow potentials on the tongue surface induced by frog glossopharyngeal efferent fiber stimulation

When the glossopharyngeal (GP) nerve of the frog was stimulated electrically, electropositive slow potentials were recorded from the tongue surface and depolarizing slow potentials from taste cells in the fungiform papillae. The amplitude of the slow potentials was stimulus strength- and the frequency-dependent. Generation of the slow potentials was not related to antidromic activity of myelinated afferent fibers in the GP nerve, but to orthodromic activity of autonomic post-ganglionic C fibers in the GP nerve. Intravenous injection of atropine abolished the positive and depolarizing slow potentials evoked by GP nerve stimulation, suggesting that the slow potentials were induced by the activity of parasympathetic post-ganglionic fibers. The amplitude and polarity of the slow potentials depended on the concentration of adapting NaCl solutions applied to the tongue surface. These results suggest that the slow potentials recorded from the tongue surface and taste cells are due to the liquid junction potential generated between saliva secreted from the lingual glands by GP nerve stimulation and the adapting solution on the tongue surface.     

Prion infection of skeletal muscle cells and papillae in the tongue

The presence of the prion agent in skeletal muscle is thought to be due to the infection of nerve fibers located within the muscle. We report here that the pathological isoform of the prion protein, PrP(Sc), accumulates within skeletal muscle cells, in addition to axons, in the tongue of hamsters following intralingual and intracerebral inoculation of the HY strain of the transmissible mink encephalopathy agent. Localization of PrP(Sc) to the neuromuscular junction suggests that this synapse is a site for prion agent spread between motor axon terminals and muscle cells. Following intracerebral inoculation, the majority of PrP(Sc) in the tongue was found in the lamina propria, where it was associated with sensory nerve fibers in the core of the lingual papillae. PrP(Sc) staining was also identified in the stratified squamous epithelium of the lingual mucosa. These findings indicate that prion infection of skeletal muscle cells and the epithelial layer in the tongue can be established following the spread of the prion agent from nerve terminals and/or axons that innervate the tongue. Our data suggest that ingestion of meat products containing prion-infected tongue could result in human exposure to the prion agent, while sloughing of prion-infected epithelial cells at the mucosal surface of the tongue could be a mechanism for prion agent shedding and subsequent prion transmission in animals.     

Selectivity of lingual nerve fibers to chemical stimuli

The cell bodies of the lingual branch of the trigeminal nerve were localized in the trigeminal ganglion using extracellular recordings together with horseradish peroxidase labeling from the tongue. Individual lingual nerve fibers were characterized with regard to their conduction velocities, receptive fields, and response to thermal, mechanical, and chemical stimuli. Fibers were classified as C, A delta, A beta, cold, and warm. The chemical stimuli included NaCl, KCl, NH4Cl, CaCl2, menthol, nicotine, hexanol, and capsaicin. With increasing salt concentration the latency of the response decreased and the activity increased. The responses elicited by salts (to 2.5 M), but not nonpolar stimuli such as menthol, were reversibly inhibited by 3.5 mM of the tight junction blocker, LaCl3. These data suggest that salts diffuse into stratified squamous epithelia through tight junctions in the stratum corneum and stratum granulosum, whereupon they enter the extracellular space. 11 C fibers were identified and 5 were characterized as polymodal nociceptors. All of the C fibers were activated by one or more of the salts NaCl, KCl, or NH4Cl. Three C fibers were activated by nicotine (1 mM), but none were affected by CaCl2 (1 M), menthol (1 mM), or hexanol (50 mM). However, not all C fibers or even the subpopulation of polymodals were activated by the same salts or by nicotine. Thus, it appears that C fibers display differential responsiveness to chemical stimuli. A delta fibers also showed differential sensitivity to chemicals. Of the 35 characterized A delta mechanoreceptors, 8 responded to NaCl, 9 to KCl, 9 to NH4Cl, 0 to CaCl2, menthol, or hexanol, and 2 to nicotine. 8 of 9 of the cold fibers (characterized as A delta''s) responded to menthol, none responded to nicotine, 8 of 16 were inhibited by hexanol, 9 of 19 responded to 2.5 M NH4Cl, 5 of 19 responded to 2.5 M KCl, and 1 of 19 responded to 2.5 M NaCl. In summary, lingual nerve fibers exhibit responsiveness to chemicals introduced onto the tongue. The differential responses of these fibers are potentially capable of transmitting information regarding the quality and quantity of chemical stimuli from the tongue to the central nervous system.     

Refinement of innervation accuracy following initial targeting of peripheral gustatory fibers

During development, axons of the chorda tympani nerve navigate to fungiform papillae where they penetrate the lingual epithelium, forming a neural bud. It is not known whether or not all chorda tympani axons initially innervate fungiform papillae correctly or if mistakes are made. Using a novel approach, we quantified the accuracy with which gustatory fibers successfully innervate fungiform papillae. Immediately following initial targeting (E14.5), innervation was found to be incredibly accurate: specifically, 94% of the fungiform papillae on the tongue are innervated. A mean of five papillae per tongue were uninnervated at E14.5, and the lingual tongue surface was innervated in 17 places that lack fungiform papillae. To determine if these initial errors in papillae innervation were later refined, innervation accuracy was quantified at E16.5 and E18.5. By E16.5 only two papillae per tongue remained uninnervated. Innervation to inappropriate regions was also removed, but not until later, between E16.5 and E18.5 of development. Therefore, even though gustatory fibers initially innervate fungiform papillae accurately, some errors in targeting do occur that are then refined during later embryonic periods. It is likely that trophic interactions between gustatory neurons and developing taste epithelium allow appropriate connections to be maintained and inappropriate ones to be eliminated.     

Non-synaptic transformation of gustatory receptor potential by stimulation of the parasympathetic fiber of the frog glossopharyngeal nerve

When the glossopharyngeal nerve (GP) in the frog was strongly stimulated electrically, slow potentials were elicited from the tongue surface and taste cells in the fungiform papillae. Injection of atropine completely blocked these slow potentials. The present and previous data indicate that the slow potentials induced in the tongue surface and taste cells are due to a liquid junction potential between saliva secreted from the lingual glands due to parasympathetic fiber activity and an adapting solution on the tongue surface. Intracellularly recorded depolarizing receptor potentials in taste cells induced by 0.5 M NaCl and 3 mM acetic acid were enhanced by depolarizing slow potentials induced by GP nerve stimulation, but were depressed by the hyperpolarizing slow potentials. On average, the receptor potential of taste cells for 0.5 M NaCl was increased by 25% by the GP nerve-induced slow potential, but the receptor potential of taste cells for 3 mM acetic acid was decreased by 1% by the slow potential. These transformations of receptor potentials in frog taste cells were not due to a synaptic event initiated between taste cells and the efferent nerve fiber, but due to a non-synaptic event, a lingual junction potential generated in the dorsal lingual epithelium by GP nerve stimulation.     

Taste cell responses in the frog are modulated by parasympathetic efferent nerve fibers

We studied the anatomical properties of parasympathetic postganglionic neurons in the frog tongue and their modulatory effects on taste cell responses. Most of the parasympathetic ganglion cell bodies in the tongue were found in extremely small nerve bundles running near the fungiform papillae, which originate from the lingual branches of the glossopharyngeal (GP) nerve. The density of parasympathetic postganglionic neurons in the tongue was 8000-11,000/mm(3) of the extremely small nerve bundle. The mean major axis of parasympathetic ganglion cell bodies was 21 microm, and the mean length of parasympathetic postganglionic neurons was 1.45 mm. Electrical stimulation at 30 Hz of either the GP nerve or the papillary nerve produced slow hyperpolarizing potentials (HPs) in taste cells. After nicotinic acetyl choline receptors on the parasympathetic ganglion cells in the tongue had been blocked by intravenous (i.v.) injection of D-tubocurarine (1 mg/kg), stimulation of the GP nerve did not induce any slow HPs in taste cells but that of the papillary nerve did. A further i.v. injection of a substance P NK-1 antagonist, L-703,606, blocked the slow HPs induced by the papillary nerve stimulation. This suggests that the parasympathetic postganglionic efferent fibers innervate taste cells and are related to a generation of the slow HPs and that substance P is released from the parasympathetic postganglionic axon terminals. When the resting membrane potential of a taste cell was hyperpolarized by a prolonged slow HP, the gustatory receptor potentials for NaCl and sugar stimuli were enhanced in amplitude, but those for quinine-HCl and acetic acid stimuli remained unchanged. It is concluded that frog taste cell responses are modulated by activities of parasympathetic postganglionic efferent fibers innervating these cells.     

Light and scanning electron microscopic study of the tongue in the cormorant Phalacrocorax carbo (Phalacrocoracidae, Aves)

The tongue of the cormorant Phalacrocorax carbo is a small, immobile structure with a length of 1.4 cm, situated in the middle part of the elongated lower bill. The uniquely shaped tongue resembles a mushroom, with a short base and an elongated dorsal part with sharpened anterior and posterior tips. A median crest can be observed on the surface of the tongue. Examination by light and scanning electron microscopy revealed that the whole tongue is formed by a dense connective tissue with many bundles of elastic fibers. The lingual mucosa is covered by a multilayered keratinized epithelium. The thickest, horny layer of the lingual epithelium was observed on the surface of the median crest and on the posterior tip of the tongue. Lingual glands are absent in cormorants. The framework of the tongue is composed of a hyoid cartilage incorporated into the base. The localization and structure of the tongue in the cormorant show that it is a rudimentary organ and that the lingual body, usually well-developed in birds, is conserved.     

c-fos expression in trigeminal spinal nucleus after electrical stimulation of the hypoglossal nerve in the rat

The expression of the immediate early gene, c-fos, was used to determine the distribution of brainstem neurons activated by stimulation of the distal hypoglossal nerve (XIIn) trunk. The traditional view of the XIIn is one of purely motor function; however, stimulation of XIIn excites neurons in the trigeminal spinal nucleus. The rationale for this study was to use c-fos expression as a marker for postsynaptic activity to define the pattern of brainstem neurons excited by XIIn stimulation. It was further hypothesized that if the afferent fibers that course within XIIn supply deep lingual tissues, then c-fos expression after direct stimulation of XIIn should display a pattern similar to that seen after chemical irritant stimulation of the deep tongue muscle. In barbiturate-anesthetized male rats electrical stimulation of XIIn produced a significant increase in Fos-positive neurons in the dorsal paratrigeminal nucleus (dPa5) and laminae I-II of caudal subnucleus caudalis (Vc) and upper cervical dorsal horn. Mustard oil injection into the deep tongue muscle also produced an increase in c-fos expression in dPa5; however, the highest density of expression occurred in laminae I-II at the dorsomedial aspect of rostral Vc. Both electrical stimulation of XIIn and mustard oil stimulation of the deep tongue increased c-fos expression in the caudal ventrolateral medulla, an autonomic relay nucleus. These results suggest that one site of innervation for afferent fibers that travel within the distal trunk of XIIn is to supply the deep tongue muscle and to terminate in the dPa5. A second group of postsynaptic neurons activated only by XIIn stimulation was located in lamina I-II in caudal portions of Vc and upper cervical dorsal horn, a laminar distribution consistent with a role for XIIn afferents in sensory or autonomic aspects of lingual function.     

c-fos expression in trigeminal spinal nucleus after electrical stimulation of the hypoglossal nerve in the rat

The expression of the immediate early gene, c-fos, was used to determine the distribution of brainstem neurons activated by stimulation of the distal hypoglossal nerve (XIIn) trunk. The traditional view of the XIIn is one of purely motor function; however, stimulation of XIIn excites neurons in the trigeminal spinal nucleus. The rationale for this study was to use c-fos expression as a marker for postsynaptic activity to define the pattern of brainstem neurons excited by XIIn stimulation. It was further hypothesized that if the afferent fibers that course within XIIn supply deep lingual tissues, then c-fos expression after direct stimulation of XIIn should display a pattern similar to that seen after chemical irritant stimulation of the deep tongue muscle. In barbiturate-anesthetized male rats electrical stimulation of XIIn produced a significant increase in Fospositive neurons in the dorsal paratrigeminal nucleus (dPa5) and laminae I-II of caudal subnucleus caudalis (Vc) and upper cervical dorsal horn. Mustard oil injection into the deep tongue muscle also produced an increase in c-fos expression in dPa5; however, the highest density of expression occurred in laminae I-II at the dorsomedial aspect of rostral Vc. Both electrical stimulation of XIIn and mustard oil stimulation of the deep tongue increased c-fos expression in the caudal ventrolateral medulla, an autonomic relay nucleus. These results suggest that one site of innervation for afferent fibers that travel within the distal trunk of XIIn is to supply the deep tongue muscle and to terminate in the dPa5. A second group of postsynaptic neurons activated only by XIIn stimulation was located in lamina I-II in caudal portions of Vc and upper cervical dorsal horn, a laminar distribution consistent with a role for XIIn afferents in sensory or autonomic aspects of lingual function.     

Histoarchitectonics of the cholinergic innervation of the frog tongue

Acetylcholinesterase (AChE) activity was studied in dorsal tongue surface structures and in both tongue nerves (hypoglossal and glossopharyngeal) of frogs (Rana temporaria). AChE was found in nerve fibers of fungiform and filiform papillae, blood vessels, glandular ducts of tongue mucosa, both nerve fibers and also in the bodies of cholinergic neurons in subepithelial connective tissue and along the glossopharyngeal nerve. Their parasympathetic origin was suggested. The experiments with butirilthiocholin have revealed no activity of non-specific cholinesterases in the above structures. Possible role of cholinergic system in the regulation of tongue receptor function is discussed.     

Taste placodes are primary targets of geniculate but not trigeminal sensory axons in mouse developing tongue

Tongue embryonic taste buds begin to differentiate before the onset of gustatory papilla formation in murine. In light of this previous finding, we sought to reexamine the developing sensory innervation as it extends toward the lingual epithelium between E 11.5 and 14.5. Nerve tracings with fluorescent lipophilic dyes followed by confocal microscope examination were used to study the terminal branching of chorda tympani and lingual nerves. At E11.5, we confirmed that the chorda tympani nerve provided for most of the nerve branching in the tongue swellings. At E12.5, we show that the lingual nerve contribution to the overall innervation of the lingual swellings increased to the extent that its ramifications matched those of the chorda tympani nerve. At E13.0, the chorda tympani nerve terminal arborizations appeared more complex than those of the lingual nerve. While the chorda tympani nerve terminal branching appeared close to the lingual epithelium that of the trigeminal nerve remained rather confined to the subepithelial mesenchymal tissue. At E13.5, chorda tympani nerve terminals projected specifically to an ordered set of loci on the tongue dorsum corresponding to the epithelial placodes. In contrast, the lingual nerve terminals remained subepithelial with no branches directed towards the placodes. At E14.5, chorda tympani nerve filopodia first entered the apical epithelium of the developing fungiform papilla. The results suggest that there may be no significant delay between the differentiation of embryonic taste buds and their initial innervation.     

Deglutitive movement of the tongue under local anesthesia

The purpose of the present study was to investigate whether or not sensory input from the tongue affects deglutitive tongue movement. Subjects were seven healthy volunteers with anesthetic applied to the surface of the tongue (surface group) and seven healthy volunteers with the lingual nerve blocked by anesthetic (blocked group). We established six stages in deglutition and analyzed deglutitive tongue movement and the time between the respective stages by cineradiography before and after anesthesia. After anesthesia in both surface and blocked groups, deglutitive tongue movement slowed and bolus movement was delayed. The deglutitive tongue tip retreated in the blocked group. These results suggest that delay of tongue movement by anesthesia causes weak bolus propulsion and that deglutitive tongue tip position is affected by sensory deprivation of the tongue or the region innervated by the inferior alveolar nerve.     

Morphofunctional characteristics of the accessory structures of the cat lingual receptors

By means of scanning electron microscopy detailed topographic map of the cat tongue was made. It was demonstrated that besides three types of chemoreceptor formations, the dorsal surface of the cat tongue possess the same amount of additional mechanoreceptor structures. By means of registration of afferent impulses, from the lingual nerve fibres at their adequate stimulation, it was possible to determine functional role of some of them.     

Effects of antidromic activity in gustatory nerve fibers on taste disc cells of the frog tongue

Summary Antidromic electrical stimulation of the lingual branch of the glossopharyngeal (IX) nerve of the frog was carried out while recording intracellular potentials of taste disc cells.Antidromic activation of sensory fibers resulted in depolarization of cells of the upper layer of the disc and most commonly in hyperpolarization of the cells in the lower layer. These changes in potential exhibited latencies greater than 1 s (Fig. 3), and thus cannot be due to electrotonic effects of action potentials in terminals of IX nerve fibers, which have much shorter conduction times. These cell potentials also showed summation, adaptation and post-stimulus rebound (Figs. 3, 4).Depending upon the chemical stimulus used, antidromic activity produced either depression or enhancement of gustatory fiber discharge in response to taste stimuli (Fig. 5).Alteration of the resting membrane potential by current injection did not significantly modify the antidromically evoked potentials (Fig. 8), whereas chemical stimulation of the tongue did (Fig. 7), indicating that these potential changes are not the result of passive electrical processes.These experimental results indicate that the membrane potential of taste disc cells can be modified by antidromic activity in their afferent nerves. This mechanism may be responsible for peripheral interactions among gustatory units of the frog tongue.The research was supported in part by NIH grant NS-09168.     

Patterns of motor activity in the isolated nerve cord of the octopus arm

The extremely flexible octopus arm provides a unique opportunity for studying movement control in a highly redundant motor system. We describe a novel preparation that allows analysis of the peripheral nervous system of the octopus arm and its interaction with the muscular and mechanosensory elements of the arm's intrinsic muscular system. First we examined the synaptic responses in muscle fibers to identify the motor pathways from the axial nerve cord of the arm to the surrounding musculature. We show that the motor axons project to the muscles via nerve roots originating laterally from the arm nerve cord. The motor field of each nerve is limited to the region where the nerve enters the arm musculature. The same roots also carry afferent mechanosensory information from the intrinsic muscle to the axial nerve cord. Next, we characterized the pattern of activity generated in the dorsal roots by electrically stimulating the axial nerve cord. The evoked activity, although far reaching and long lasting, cannot alone account for the arm extension movements generated by similar electrical stimulation. The mismatch between patterns of activity in the isolated cord and in an intact arm may stem from the involvement of mechanosensory feedback in natural arm extension.     

Distribution of calcitonin gene-related peptide and substance P-immunoreactive nerve fibers and their correlation in the periodontal ligament of the mouse incisor.

The distribution of calcitonin gene-related peptide (CGRP)- and substance P (SP)-immunoreactive (IR) nerve fibers and their correlation in the periodontal ligament of mouse incisors were examined by indirect immunofluorescence. Both CGRP-IR and SP-IR thin nerve fibers were abundant in the apical and middle third of the periodontal ligament. In the lingual portion of the incisal periodontal ligament, these nerve fibers were localized in the alveolar half of the periodontal ligament and were observed as free nerve endings. No CGRP-IR and SP-IR specialized nerve endings, such as Ruffini-like corpuscles, were observed. In the labial periodontal ligament, CGRP-IR and SP-IR nerve fibers ran along the incisal axis. The distribution of CGRP-IR nerve fibers was very similar to that of SP-IR nerve fibers.     

Ultrastructural study of the precursor to fungiform papillae prior to the arrival of sensory nerves in the fetal rat.

The structure of precursors to fungiform papillae without taste buds, prior to the arrival of sensory nerve fibers at the papillae, was examined in the fetal rat on embryonic day 13 (E13) and 16 (E16) by light and transmission electron microscopy in an attempt to clarify the mechanism of morphogenesis of these papillae. At E13, a row of rudiments of fungiform papillae was arranged along both sides of the median sulcus of the lingual dorsal surface, and each row consisted of about 10 rudiments. There was no apparent direct contact between papillae rudiments and sensory nerves at this time. Bilaterally towards the lateral side of the tongue, adjacent to these first rudiments of fungiform papillae, a series of cord-like invaginations of the dorsal epithelium of the tongue into the underlying connective tissue, representing additional papillary primordia parallel to the first row, was observed. The basal end of each invagination was enlarged as a round bulge, indented at its tip by a mound of fibroblasts protruding into the bulge. At E16 there was still no apparent direct contact between rudiments of fungiform papillae and sensory nerves. Each rudiment apically contained a spherical core of aggregating cells, which consisted of a dense assembly of large, oval cells unlike those in other areas of the lingual dorsal epithelium. The differentiation of these aggregated cells was unclear. The basal lamina was clearly recognizable between the epithelium of the rudiment of fungiform papillae and the underlying connective tissue. Spherical structures, which appeared to be sections of the cord-like invaginations of the lingual epithelium that appeared on E13, were observed within the connective tissue separated from the dorsal lingual epithelium. Transverse sections of such structures revealed four concentric layers of cells: a central core, an inner shell, an outer shell, and a layer of large cells. Bundles of fibers were arranged in the central core, and the diameters of bundles varied somewhat depending on the depth of the primordia within the connective tissue and their distance from the median sulcus. Ultrastructural features of cells in the outer shell differed significantly in rudiments close to the lingual epithelium as compared to those in deeper areas of connective tissue. Around the outer shell there was a large-cell layer consisting of one to three layers of radially elongated, oval cells that contained many variously sized, electron-dense, round granules. Large numbers of fibroblasts formed dense aggregates around each spherical rudiment, and were separated by the basal lamina from the large-cell epithelial layer. Progressing from deep-lying levels of the rudiments of the papillae to levels close to the lingual surface epithelium, the central core, inner shell, and outer shell gradually disappeared from the invaginated papillary cords.     

Occurrence of parasympathetic vasodilator fibers in the lower lip of the guinea-pig

The present study was designed to examine whether there are parasympathetic vasodilator fibers in the lower lip of the guinea-pig. Electrical stimulation of the central cut end of the lingual nerve of guinea-pigs evoked intensity- and frequency-dependent decreases in lower lip blood flow and systemic arterial blood pressure (SABP). Pretreatment with guanethidine, a postganglionic sympathetic nerve blocker and antihypertensive drug (30 mg kg⁻¹, s.c., 24 h prior to experiments), reduced the magnitude of the decrease in SABP while the intensity- and frequency-dependent increases of the lip blood flow occurred by the lingual nerve stimulation only on the side ipsilateral to stimulation. Increases in the lip blood flow evoked by lingual nerve stimulation in guanethidine pretreated guinea-pigs were reduced by hexamethonium (an autonomic ganglion cholinergic blocker) in a dose-dependent manner. When fluoro-gold (a retrograde neural tracer) was injected into the lower lip, labeled neurons were observed in the ipsilateral otic ganglion. The present study indicates the presence of parasympathetic vasodilator fibers originating from the otic parasympathetic ganglion in the guinea-pig lower lip, similar to those reported previously in rats, cats, rabbits and humans.