Low-Threshold Mechanoreceptive Afferents in the Human Lingual Nerve

Mats Trulsson1 and Gregory K. Essick2

1 Department of Physiology, Umeå University, S-901 87 Umeå, Sweden; and 2 Department of Prosthodontics and Curriculum in Neurobiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7450

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Trulsson, Mats and Gregory K. Essick. Low-threshold mechanoreceptive afferents in the human lingual nerve. J. Neurophysiol. 77: 737-748, 1997. Intrafascicular multiunit activity and impulses in single mechanoreceptive afferents were recorded from the human lingual nerve with permucosally inserted tungsten microelectrodes. Nylon filaments and blunt glass probes were used for mechanical stimulation of the mucosa of the dorsal surface of the tongue. The innervation territories of nine nerve fascicles were mapped during multiunit recordings. All fascicle fields included the tip of the tongue, suggesting a particularly high innervation density for this area. Thirty-three single mechanoreceptive afferents were isolated and studied. Of these afferents, 22 were characterized by very small mucosal receptive fields (range: 1-19.6 mm2; geometric mean: 2.4 mm2) and responded to extremely low mechanical forces (force threshold range: 0.03-2 mN; geometric mean: 0.15 mN). As such, it was concluded that these "superficial" units terminated near the surface of the tongue. The remaining 11 units responded to probing of large areas of the tongue (>200 mm2) and exhibited high force thresholds (>= 4 mN). It was concluded that these "deep" units terminated in the muscle mass of the tongue. Fourteen of the superficial units were classified as rapidly adapting and resembled the fast-adapting type I afferents described for the glabrous skin of the human hand. The rapidly adapting units responded both during the application and removal of, but not during maintenance of, the mechanical stimuli on the receptive field. Two types of slowly adapting responses were observed. One type (characteristic of only 2 units) was characterized by a pronounced sensitivity to force change during the application and removal of the mechanical stimuli and an irregular static discharge during maintenance of the stimulus on the receptive field. In contrast, the other six units exhibited a weak sensitivity to force change, a highly regular static discharge, and spontaneous activity. As such, these two types of slowly adapting units resembled the slowly adapting I and II afferents, respectively, described for the hand. All 11 deep units were slowly adapting, and 7 were, in addition, spontaneously active. The units were not equally sensitive to the application and removal of the mechanical stimuli, suggesting at least two different modes of termination in tongue muscle. The deep units reliably encoded information about tongue movements in the absence of direct contact with the receptive field. In contrast, the superficial units responded vigorously when the tongue was moved to bring the receptive field into physical contact with other intraoral structures.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The technique of microneurography allowing multi- and single-unit recordings to be made from peripheral nerves of human subjects was first described by Vallbo and Hagbarth (1968; see also Torebjörk et al. 1987; Vallbo et al. 1979). In the extremities, recordings have been obtained to study the function of skin and muscle mechanoreceptive afferents, nociceptors, thermoreceptors, sympathetic fibers, and skeleto- and fusimotor fibers (Johansson and Vallbo 1983; Vallbo et al. 1979; Wallin and Fagius 1988). The first recordings from the trigeminal afferent system in humans were reported in 1976 (Johansson and Olsson 1976). Since then, a number of studies on mechanoreceptive afferents innervating facial skin and periodontal ligament has been published (e.g., Edin et al. 1995; Furusawa et al. 1992; Johansson et al. 1988a,b; Nordin 1990; Nordin and Hagbarth 1989; Trulsson 1993; Trulsson and Johansson 1994, 1996; Trulsson et al. 1992). Except for periodontal mechanoreceptors, which have been described in some detail (e.g., see Trulsson and Johansson 1996), very little is known about the functional properties of mechanoreceptors innervating tissues inside the oral cavity (Furusawa et al. 1992; Johansson et al. 1988b). The aim of the present study was to further explore the feasibility of the microneurographic technique as a tool to investigate the low-threshold mechanoreceptive innervation of human intraoral tissues. The anterior portion of the tongue, innervated by the lingual nerve, was chosen for study given its salient contributions to orofacial function. An abstract of preliminary results has been published previously (Trulsson et al. 1995).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Subjects and general procedure

The results presented in this paper are based on data obtained from eight healthy subjects (2 males, 6 females, ages 20-32 yr) who participated in one or more recording sessions lasting 3-4 h each. After informed consent was obtained, the subject was comfortably seated in a dental chair. A trimmed thermoplastic block was placed between the upper and lower molars of one side to keep the mouth open in a stable position. A horizontal plate attached to the thermoplastic block restricted upward movement of the tongue and prevented contact between the tongue and electrode. Because the plate covered only the middle third of the tongue, the anterior third and tip remained free for manipulation. Saliva was evacuated as needed from the mouth with the use of a portable suction system. A chart was placed in the subject's reach on which the subject could indicate the occurrence of altered sensations ("paresthesia," "pain," etc.) during microelectrode insertion and manipulation. Clinical asepsis was maintained. The study was approved by the Committee on Investigations Involving Human Subjects at the University of North Carolina School of Dentistry.

Neurophysiological recording technique

The electrophysiological recording technique and manual handling of the microelectrode were similar to that employed to study the human inferior alveolar nerve (Johansson and Olsson 1976; Trulsson 1993; Trulsson and Johansson 1994, 1996; Trulsson et al. 1992). Briefly summarized, either the right or left lingual nerve was approached intraorally with an 0.2-mm-diam tungsten needle electrode (Fig. 1, left). The distal 1 mm of the electrode tapered to a 5-µm tip. The electrode was coated with lacquer to within 10-30 µm of the tip, which provided an impedance, measured in situ, of 100-700 kOmega at 1 kHz. Neural signals were amplified (×10,000; bandwidth 0.47-5 kHz) by a probe fixed with a headband to one side of the subject's head.


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FIG. 1. Illustration of permucosal microneurography to study the lingual nerve in humans. Note the general intraoral position of the tungsten microelectrode, indicated on the tracing of the jaw and tongue seen from the midsagittal plane (left). Loads were applied to the lingual mucosa by a hand-held probe equipped with a nylon filament and strain gauges (SG) for continuous force (F) measurement (right).

To guide insertion of the electrode during search for the lingual nerve, electrical stimulation (square-wave pulses of negative polarity, 0.2 ms in duration, 0.1-1 mA) was delivered intermittently through the electrode. Paresthesias in the tongue indicated that the electrode approximated the nerve. After the nerve was impaled, multiunit activity from a large number of afferents was frequently observed and recorded. To evoke afferent activity, the subject was asked to move the anterior part of the tongue back and forth under the horizontal restraint plate. Alternatively, the experimenter mechanically stimulated the mucosa intermittently.

To study individual afferents, the recording electrode was manipulated in minute steps until impulse responses were isolated from a single afferent. The electrode was left in this recording position, supported only by the surrounding tissue. Stable single-unit recordings were so obtained for 10-20 min. The small electrode movements occasionally evoked short-lasting and localized paresthesias in the tongue. However, no persistent paresthesia or other complication was reported by any subject after the recording sessions.

Mechanical stimulation

A set of calibrated nylon monofilaments (0.03, 0.06, 0.12, 0.25, 0.5, 1, 2, 4, 8, and 16 mN) was used to apply mechanical stimuli by hand to the lingual mucosa. In addition, the prodding force could be measured with a special set of two nylon filaments (5 and 50 mN) mounted at right angles to a bar device equipped with strain gauges (DC-120 Hz; Fig. 1, right). As such, the strain gauges provided a continuous signal of the force applied perpendicular to the mucosal surface. Local taps and more forceful indentations of the mucosa were manually delivered by blunt glass probes.

Unit characterization and classification

After the electrical response from each single unit was isolated, the calibrated filaments were used to identify the location on the tongue surface at which the afferent was most sensitive. This site was marked and the threshold force was determined. The threshold force was defined as the calibrated force of the least stiff filament that evoked a response on half of the stimulus trials. For spontaneously active units, the threshold was defined as the force of the least stiff filament that produced a clear modulation of the ongoing activity. After threshold determination, the receptive field was identified by carefully mapping the mucosal area over which the filament that provided 4 times the threshold force evoked a response (Johansson and Vallbo 1980). For units with a threshold >= 8 mN, the receptive field was mapped by use of a blunt glass probe.

Receptor adaptation was evaluated by the maintained application for >= 2 s of a filament that delivered >= 4 times the threshold force. Units that exhibited sustained discharge for the duration of the stimulation were classified as slowly adapting (SA), and otherwise as rapidly adapting (RA). Additionally, units were classified as spontaneously active if they exhibited an ongoing background discharge in the absence of external stimulation. On the basis of thresholds to mechanical stimulation and receptive field size, each unit was classified as "superficial" or "deep." The superficial units with SA responses were further subcategorized on the basis of their regularity of discharge in response to maintained application of the mechanical stimuli.

Recording of tongue movements

For single units that remained isolated after characterization of their response properties to externally applied stimuli, the subject was asked to carefully protrude and retract the tongue (excursions ~10 mm in amplitude). The phase of the rhythmic horizontal tongue movements was recorded with the use of the strain-gauge-equipped filament (see above), which the experimenter held close to the tip of the tongue. During protrusion, the nylon filament buckled in response to the pressure applied by the tongue, thereafter providing a relatively constant signal. During retraction, the filament became unloaded, returning the force signal to baseline.

Data collection and processing

The nerve and force signals were displayed simultaneously on a computer screen during the recording session, and sampled at 12.8 kHz and 800 Hz, respectively (12-bit resolution) with the use of a flexible data collection/analysis computer system (SC/ZOOM, Department of Physiology, Umeå University). Individual action potentials were identified off-line with the use of previously described algorithms (Edin et al. 1988) implemented by the SC/ZOOM system. All spikes were visually examined on an expanded time scale before they were accepted as representing unitary activity. The instantaneous discharge rate was calculated as the inverse of the time interval between consecutive action potentials.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Multiunit recordings

Innervation territories of nine nerve fascicles were mapped during multiunit recording (cf. Hagbarth et al. 1970). Each territory was defined as the mucosal area from which detectable mass responses could be evoked during gentle stroking with a blunt glass probe. Figure 2 shows the relative size and location of the nine innervation territories. Note that the posterior border could not be determined for some of the fields because they extended under the horizontal restraint plate.


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FIG. 2. Innervation territories of 9 nerve fascicles in the lingual nerve mapped in 6 subjects. Shaded areas surrounded by heavy lines: innervation territories on the dorsal side of the tongue. A single discontinuity in the heavy line indicates that the border could not be determined. For the 4 fields that extended to the ventral surface of the tongue, the border is indicated by dashed lines.

Three observations suggested that the multiunit responses were recorded from a large number of low-threshold mechanoreceptive afferents within the impaled fascicle, but not from fibers in other fascicles (see also Hagbarth et al. 1970). First, careful movements of the electrode caused little or no change in the responsive area until its borders suddenly changed, indicating that the tip of the electrode had slipped into a new fascicle. Second, at any electrode position within a fascicle, a multiunit response could be recorded whenever a weak tactile stimulus was applied within the responsive area. And third, the extent of the responsive area was not influenced appreciably by changes in the prodding force.

Most strikingly, the fascicle fields always included the tip of the tongue. Many of the fields extended at least 3 cm posteriorally, but no field extended more than 1-2 mm across the midline. Four of the fields also innervated the ventral surface of the tongue, i.e., the fields extended below the peripheral border of the tongue (indicated by a dashed line in Fig. 2).

Multiunit recordings exhibited little activity in the absence of externally applied stimulation. In response to mucosa deformation, the response to the application and removal of the stimuli strongly predominated over the response to maintained loading: distinct ON and OFF discharges were observed at the onset and offset of stimulus application, respectively. Moreover, particularly vigorous responses were elicited by mechanical stimuli that moved across the surface of the receptive area.

Single-unit recordings

Impulse responses were recorded from 33 single mechanoreceptive afferents in the lingual nerve. For 22 of these afferents, distinct receptive fields could be identified on the dorsal mucosa of the anterior part of the tongue. These units were characterized by small receptive fields (<20 mm2) and low force thresholds (<= 2 mN). The terminal endings of these units were judged to be situated superficially in the tongue, and the afferents were consequently labeled superficial. The remaining 11 units responded to probing of large areas of the tongue (>200 mm2) and exhibited force thresholds (>= 4 mN) higher than those of any of the superficial units. Because these observations suggested that their terminal endings were situated deeply in the tongue muscle, these 11 afferents were labeled deep.

SUPERFICIAL UNITS. RA units. Of the 22 superficial units, 14 were classified as RA. Their receptive fields consisted of a small, approximately circular or oval, well-defined area of high and relatively uniform sensitivity (see black areas in Fig. 3A). The receptive fields of three units were found to possess more than one zone of maximal sensitivity during stimulation with nylon filaments. The size of the receptive fields for the RA units ranged between 1 and 12.5 mm2 (geometric mean: 2.0 mm2; n = 14 units; Fig. 3B). None of the units were activated by taps applied to sites remote from the receptive field, indicating that the sample did not contain Pacinian corpuscle afferents (cf. Hunt and McIntyre 1960; Johansson and Vallbo 1979).


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FIG. 3. Receptive field characteristics of 22 superficial mechanoreceptive afferents located in the human lingual nerve. A: schematic drawing of the tongue illustrates the locations and relative sizes of the receptive fields of the 3 different mechanoreceptor classes [rapidly adapting (RA), slowly adapting (SA) irregular, and SA regular]. B and C: bars indicate geometric mean receptive field area (B) and geometric mean threshold force (C) for each class of mechanoreceptors. Vertical bars: measn ± SE. For comparison, symbols indicate geometric mean receptive field area (B) and geometric mean threshold force (C) for cutaneous mechanoreceptive afferents studied by Edin et al. (1995) from the human median (black-square), radial (black-triangle), and mental (bullet ) nerves. For these latter data, vertical bars indicate SE.

All RA units exhibited burst responses at the beginning and end of a maintained indenting stimulus. Examples of such ON and OFF responses from two different units are shown in Fig. 4, A and B. The OFF response was occasionally greater than the ON response, which reflected, in part, the negative force exerted by the nylon filament when removed from the densely packed filiform papillae (Fig. 4, A and B, north-west-arrow ). Application of graded stimuli revealed that stiffer filaments produced stronger responses. The maximum discharge frequencies obtained in response to sudden tissue displacement were on the order of 200-300 imp/s. The threshold as determined with the calibrated filaments ranged between 0.03 and 0.5 mN (geometric mean: 0.11 mN; Fig. 3C). The responses from two units with small receptive fields (<2 mm2) near the tip of the tongue exhibited an ongoing background discharge that was not spontaneous, but clearly time-locked to the arterial pulse (Fig. 4C). Pulse-synchronous discharge has been observed previously in cutaneous mechanoreceptors located in the infraorbital (cf. Fig. 6B of Nordin and Hagbarth 1989) and mental nerves (M. Trulsson, G. K. Essick, and B. B. Edin, unpublished data).


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FIG. 4. Example of recordings from 3 RA superficial units in the lingual nerve. A: data from a single unit displaying a 3-mm2 receptive field at the tip of the tongue (threshold force: 0.06 mN). B: data from a single unit responding to forces applied to a single filiform papilla at the tip of the tongue (threshold force: 0.03 mN). In A and B, top traces illustrate the force applied by the nylon filament; middle and bottom traces depict the instantaneous discharge frequency and the sampled neurogram, respectively. Note that the OFF response appears to be generated, in part, by the negative force exerted by removal of the stimulus filament from the mucosa (north-west-arrow ). C: data from a single unit displaying an oval receptive field (2 mm2) located near the midline of the tongue. The background discharge activity (shown) was not spontaneous, but time-locked to the arterial pulse. Top trace: instantaneous discharge frequency. Bottom trace: neurogram.


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FIG. 6. Example of recordings from 2 deep units in the human lingual nerve. Tracings as in Fig. 4. A: data from an SA spontaneously active unit characterized by a strong ON response and a clearly visible, but subtle, OFF response. Force threshold at most sensitive site was relatively high, 4 mN. B: data from a 2nd SA spontaneously active unit, but with a weak response to the application and no response to the removal of the stimulus (force threshold: 8 mN). A and B, right: schematic illustrations of receptive fields. Dot: point of maximal sensitivity. Heavy line: approximate area over which unit responded to perpendicular stimulation of the dorsal lingual mucosa. Dashed line: extension of the border to ventral surface of tongue.

SA units. Eight of the superficial units responded with an SA discharge to a suprathreshold force applied to the receptive field. As with the RA units, the receptive fields of the SA units consisted of an approximately circular or oval well-demarcated area of high and relatively uniform sensitivity (see white and shaded areas in Fig. 3A). The fields possessed only a single zone of maximal sensitivity and their sizes ranged between 1 and 19.6 mm2 (geometric mean: 3.3 mm2; Fig. 3B). In contrast to the RA units, some of the SA units (viz., 5 of 8 units) exhibited a steady ongoing response (range among units: 6-20 imp/s) in the absence of externally applied mechanical stimuli, i.e., they were spontaneously active. All SA units exhibited a "dynamic" response to the onset of the mechanical stimulus (Fig. 5). The maximum discharge frequencies obtained in response to rapid tissue displacement were 100-500 imp/s. The maximum response rates during maintained pressure were ~40-80 imp/s. Similar to the RA afferents, increasingly stiffer nylon filament stimuli evoked stronger discharge responses. The thresholds ranged between 0.06 and 2 mN (geometric mean: 0.27 mN; Fig. 3C).


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FIG. 5. Example of recordings from 2 SA superficial units in the human lingual nerve. Tracings as in Fig. 4. A: data from an SA irregular unit with a punctate receptive field (1 mm2) and force threshold of 0.12 mN. Note pronounced ON response and the irregular discharge during maintenance of the stimulus load. B: data from an SA regular unit with a relatively large receptive field (20 mm2) and threshold of 0.25 mN. Note the ongoing discharge in the absence of an externally applied stimulus, the highly regular discharge during maintenance of the stimulus load, and the "pause" or momentary cessation of spontaneous activity after the stimulus was removed from the receptive field. A and B, right: interspike interval histograms calculated from data collected 30-60 s after application of a nylon filament. Inserted neurograms show samples of discharge activity after 30 s of maintained stimulation. Coefficients of variation of interspike intervals were 0.96 and 0.08 for the units in A and B, respectively.

Several lines of evidence suggested that the SA responses originated from two different types of afferent units. The first type (2 of 8 units) exhibited irregular responses to maintained indentation of the receptive field, and the afferents were classified accordingly as "SA irregular" units (Fig. 5A). Interspike interval histograms demonstrated a broad, positively skewed distribution for both units (see example in Fig. 5A, right). The histograms were constructed from the second half of a 60-s period of activity evoked by maintained indentation with the filament that delivered 8 times the threshold force. Moreover, coefficients of variation of interspike intervals were 0.70 and 0.96 for the two units. The two SA irregular afferents were also particularly sensitive to change in deformation of the mucosa and showed dynamic OFF discharges similar to those exhibited by the RA units. The receptive fields were very small (1 mm2 for both units; Fig. 3, A and B), with threshold forces of 0.12 and 0.25 mN (Fig. 3C).

The second type constituted the majority of the SA units (6 of 8 units). These units exhibited highly regular responses to maintained indentation of the mucosa and thus were classified as "SA regular" units (Fig. 5B). Interspike interval histograms constructed for these six units revealed narrow, normal distributions, all similar to the example shown in Fig. 5B, right. Moreover, coefficients of variation of interspike intervals ranged between 0.06 and 0.24 (geometric mean: 0.16). Thus the coefficients of variation did not overlap for the two unit types, demonstrating that they could be distinguished solely on the basis of stimulus-evoked response regularity. All but one of the SA regular units (5 of 6 units) were spontaneously active, and all discharged at lower peak rates (<150 imp/s) than did the SA irregular units (peak rates up to 500 imp/s). The receptive field size of the SA regular units ranged between 1 and 19.6 mm2 (geometric mean: 5.3 mm2; Fig. 3, A and B); their threshold forces ranged between 0.06 and 2 mN (geometric mean: 0.31 mN; Fig. 3C).

DEEP UNITS. All 11 deep units exhibited SA responses to mechanical stimulation of the mucosa (Fig. 6). Although each receptive field possessed only a single zone of maximal sensitivity, responses could be evoked from a wide surrounding area (>200 mm2) over which sensitivity fell off gradually. The full extent of the receptive field could only be mapped for six of the units, and ranged between 200 and 650 mm2. For four of these, the fields consisted of an approximately oval area with obscure boundaries on the dorsal part of the tongue (Figs. 6B and 7, B and C); for two units, the fields also involved the ventral part of the tongue (Fig. 6A).


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FIG. 7. Responses of single mechanoreceptive afferents recorded from the lingual nerve during active tongue movements. A: data from an RA superficial unit that exhibited a small receptive field on the tip of the tongue. A burst of impulses (gray bars) was elicited each time the subject moved the tongue so that the receptive field was brought into contact with a lower incisor tooth. Top trace: instantaneous discharge frequency. Bottom trace: sampled neurogram. Right: location of the receptive field on the tongue and the contact area on the tooth (shaded). B: data from an SA deep unit that responded each time the subject protruded the tongue. C: data from an SA spontaneously active deep unit that responded each time the subject retracted the tongue. B and C, top traces: solid curve indicates the phase of the rhythmic tongue movement as recorded with a strain-gauge-equipped nylon filament held close to the tip of the tongue; dashed curve (drawn by eye) estimates horizontal tongue displacement (maximum excursion ~10 mm in amplitude). Care was taken to assure that the receptive field areas (shown at right) were not contacted by other structures during the active tongue movements. Middle and bottom traces: as in A.

Seven of the deep units exhibited a steady spontaneous discharge (range among units: 8-22 imp/s). All units responded to the onset of the mechanical stimuli. Four of the units were characterized by a rather strong dynamic ON response and a visible OFF response (Fig. 6A), whereas the other seven units showed relatively weak dynamic responses (Fig. 6B). The maximum response rates to rapid skin displacement were 50-250 imp/s; those during maintained punctate pressure were ~30-70 imp/s. Force thresholds were generally high (>= 4 mN), and could not be determined for seven of the deep units because they did not respond to the stiffest calibrated filament (16 mN) available for threshold determination.

Unit responses to tongue movements

Responses were recorded from 10 superficial units (5 RA, 1 SA irregular, and 4 SA regular) and 6 deep units while the subject carefully moved the tongue outward and inward at ~0.5 cycles/s without touching the surrounding structures. In addition, attempts were made to record from three superficial units at faster rates (rates >2.0 cycles/s invariably dislocated the microelectrode). Recordings were also made from four of the superficial units (3 RA and 1 SA regular) when the subject moved the tongue outward and inward in a manner such that the receptive field of the unit contacted a lower incisor tooth.

SUPERFICIAL UNITS. In the absence of contact with other structures, the superficial units failed to respond to tongue movements, with only one exception: a spontaneously active SA regular unit with a relatively large receptive field (19.6 mm2) near the tip of the tongue. This unit responded with a few action potentials (peak frequencies: 25 imp/s) when the tongue moved in an outward direction. All superficial units that were tested, however, responded vigorously (range of peak frequencies among units: 40-100 imp/s) when the receptive field contacted a lower incisor tooth during tongue movement. Figure 7A illustrates responses from an RA unit that displayed a small receptive field (1 mm2) at the tip of the tongue. The subject moved the tongue so that the receptive field contacted the indicated tooth.

DEEP UNITS. In contrast to the superficial units, all six deep units tested exhibited modulations in discharge during tongue movements in the absence of contact with surrounding structures. Moreover, units discharged during only one of the movement phases, i.e., either when the tongue was moved outward (n = 4 units) or back inward (n = 2 units). The peak frequencies ranged from 30 to 80 imp/s among units. Figure 7, B and C, illustrates recordings from two deep units that responded when the subject moved the tongue outward and inward, respectively.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

To the authors' knowledge, peripheral neural studies involving the human tongue have been limited to intraoperative recordings from the chorda tympani in otosclerotic patients (e.g., see Diamant et al. 1963). First conducted over 30 years ago, these studies sought to obtain the summated neural response primarily to taste stimuli, but also to thermal, irritating chemical, and gross mechanical stimulation. As predicted, the mechanical stimuli were found to evoke comparatively weak responses in this predominantly gustatory nerve, which joins the lingual nerve to supply the total sensory innervation of the anterior two thirds of the tongue (cf. Girod et al. 1989; Smith and Robinson 1995).

In this paper, it is shown that it is possible to directly record, with the use of a minimally invasive technique, both multi- and single-unit responses from the lingual nerve of the fully awake and unanesthetized human subject. Moreover, for the first time, the receptive field characteristics and adaptational properties of low-threshold mechanoreceptors that supply the anterior tongue in humans are described. It was found that different mechanoreceptors exhibit properties that suggest that they terminate differentially in the tongue (superficially or deeply), and that the populations so defined possess fundamentally different capacities to encode tactile (viz., information about stimuli in contact with the mucosa) and proprioceptive (viz., information about tongue movements in the absence of physical contact with the receptive field) information. Moreover, sufficient diversity in response to simple punctate stimuli was observed within each population of afferents to argue for the existence of at least three classes of superficial units and two classes of deep units.

Our interest in addressing the low-threshold mechanoreceptive innervation of the tongue was motivated by the following. First, we had previously studied the response properties of the cutaneous mechanoreceptors that supply the midface (Johansson et al. 1988a,b) and lower face (Edin et al. 1995). Studies of intraoral mechanoreceptors represented a logical extension of this previous work. Second, the mechanoreceptive innervation of the tongue is of special note because tactile acuity on the tip of the tongue is as high as, if not higher than, that on any other body region (for review see Rath and Essick 1990). Third, clinical studies suggest that the mechanoreceptive innervation of the tongue is particularly important to normal oral sensation: Lingual-nerve-injured patients report decided improvement in oral sensation (including taste) after surgical procedures to repair this nerve, even though objective testing indicates return of somatic, but not gustatory, sensory function (Hillerup et al. 1994; see also Girod et al. 1989; Smith and Robinson 1995).

Experimental difficulties and sources of potential sample bias

A number of technical difficulties were encountered during the recording sessions. First, the shaft of the microelectrode and the connecting cable were very close to the receptive field site. This made it particularly difficult to both manipulate the position of the electrode tip and apply mechanical stimuli to the tongue. Second, inadvertent tongue movements by the subject made it difficult to precisely apply the mechanical stimuli to the receptive fields, or often dislocated the electrode. Third, the tongue became covered with saliva on a frequent basis and saliva had to be evacuated between delivery of mechanical stimuli. Because of these difficulties, many units were lost before they could be completely classified as to response properties, threshold, and receptive field size. The single units that were completely classified, and thus described in this study, afforded isolation and stable recordings for >= 10 min.

Despite the technical limitations encountered during the recording sessions, the authors do not believe that the sample of low-threshold mechanoreceptors was significantly biased as a result. For example, during the search for stable single-unit recordings, two strategies were adopted to evoke neural activity: either the subject was asked to move the tongue back and forth against the horizontal restraint plate or the experimenter mechanically stimulated the tongue. Both approaches were marred with unique problems. In the first case, the tongue movements were often transferred to the electrode; in the second case, the experimenter had to apply mechanical stimuli to the anterior part of the tongue and, at the same time, manipulate the electrode in search of single-unit recordings. Given that the deep units, but not the superficial units, were activated directly by tongue movements, it seems plausible that the choice of stimulation approach during a given recording session may have biased the sample of units obtained. However, no difference was seen in the proportions of units isolated with the two approaches. With tongue movements, 15 superficial and seven deep units were isolated; with external stimulation, 7 superficial and four deep units were isolated.

Because large-diameter fibers were more likely to be encountered than small-diameter fibers during manipulation of the electrode tip, the unit sample may be biased toward large-diameter as opposed to small-diameter low-threshold mechanoreceptive afferents. It has been shown, however, that the physiological properties of fast-conducting skin mechanoreceptive afferents are not reflected in axon size (Mackel 1988). Thus the proportions of RA and SA units, and similarly the proportions of SA irregular and SA regular units, would be expected to be the same as those observed in the absence of this sampling bias.

Of greater concern, the unit sample may be biased toward spontaneously active units because units exhibiting an ongoing, background discharge were necessarily easier to identify. Thus we cannot rule out the possibility that the deep units and the superficial SA regular units are overrepresented in the sample, because both groups exhibited a high proportion of spontaneously active units.

Superficial tongue mechanoreceptors

COMPARISON OF RESPONSE PROPERTIES WITH THOSE OF MECHANORECEPTORS IN OTHER PERIPHERAL NERVES IN HUMANS. In the present study, about two thirds of the superficial tongue units were RA and only one third were SA. Similarly, the RA units are reported to be more common in the glabrous skin of the human hand (Johansson and Vallbo 1979). In contrast, earlier studies of the hairy skin of the hand and face, of the lips, and of the oral mucosa report a predominance of SA afferents (Edin and Abbs 1991; Edin et al. 1995; Johansson and Olsson 1976; Johansson et al. 1988b; Nordin and Hagbarth 1989). For example, in the studies by Johansson et al. (1988b) and Edin et al. (1995), only about one third of mechanoreceptors supplying the facial skin and vermilion of the lip were found to be RA (the remainder were SA). Nordin and Hagbarth (1989), studying the same skin region, observed similarly that only ~10% of the mechanoreceptors were RA. Thus, in this respect, the population of low-threshold mechanoreceptors in the tongue appears to be more similar to the population of receptors in the glabrous skin of the hand than to the adjacent orofacial tissues. We interpret this finding to reflect a functional adaptation of the mechanoreceptive innervation: skin and mucosa regions that are deformed during normal physiological movements (like the hairy skin on the back of the hand, and the hairy and nonhairy skin of the face and lips) exhibit a high proportion of SA units. SA afferents, particularly the type II units, have been shown to be particularly attuned to signaling proprioceptive information (cf. Edin 1992; Johansson et al. 1988a). On the other hand, body regions that are used for explorative and manipulative behaviors (such as the glabrous skin of the hand and the mucosa of the tongue) exhibit a predominance of RA units.

In addition to a similarity with regard to proportions of RA and SA mechanoreceptors, the response properties of the superficial units in the human lingual nerve were found to be similar to those of three of the four classes of tactile afferents that innervate the glabrous skin of the human hand, i.e., fast-adapting type I (FA I) and SA type I (SA I) and type II (SA II) units (see Johansson and Vallbo 1983; Vallbo et al. 1979). On the basis of response properties identified by simple hand-held stimuli, the superficial RA units in the present sample resemble those of the FA I units. None of the superficial RA afferents, however, were activated by taps applied at sites remote to the receptive fields, indicating that the sample did not contain Pacinian corpuscle type II (FA II) afferents (cf. Hunt and McIntyre 1960; Johansson and Vallbo 1979). The absence of Pacinian afferents supplying the dorsal lingual mucosa is consistent with previous morphological (Gairns 1953; Marlow et al. 1965) and psychophysical (Verrillo 1966) studies.

With regard to the superficial SA units, those that discharged irregularly during maintained tissue deformation and exhibited a high dynamic sensitivity (the SA irregular units) clearly resemble the SA I units found in the glabrous skin of the human hand. Similarly, the SA regular units, characterized by a highly regular discharge rate, low dynamic sensitivity, and spontaneous activity, resemble the SA II units. To illustrate, the SA irregular and SA regular units of the tongue were distinguished by interspike interval histograms generated from their response to maintained mechanical stimulation. This distinction was described previously for the SA I and SA II units of the hand by Edin (1992), who observed broad, positively skewed distributions and narrow, normal distributions, respectively, for the two groups of units. The nerve endings associated with the SA I and SA II units are generally accepted to be the complex of Merkel cells and the Ruffini ending, respectively. The former has several generators of action potentials, the latter only one (Horch et al. 1974; Iggo and Muir 1969). At low forces the separate generators of the Merkel receptor independently initiate action potentials, giving rise to an irregular discharge rate.

If comparable mechanisms apply to the sensory innervation of the tongue, one can conclude that the two types of superficial SA units have different endorgans, one with multiple spike generators and one with a single generator. Current understanding of the manner in which receptors terminate in the tongue, however, does not allow the different functional classes of superficial tongue mechanoreceptors to be associated with different endorgans, as has been accomplished for the mechanoreceptors in the glabrous skin of the human hand (cf. Johansson and Vallbo 1983). Only three types of receptor endings were identified from histologic specimens from 33 human tongues by Marlow et al. (1965): free nerve endings, semiorganized coiled endings, and organized endings. All organized endings were deemed to be mucocutaneous endorgans, varying in size and shape. The semiorganized coiled ending was viewed simply as a variant of the free nerve ending. As a result of the paucity of terminal endorgans, it was concluded that "no specific receptor function can be attributed to a receptor because of its morphology" (p. 505). These findings and interpretations, however, are not in total agreement with those of earlier investigators (e.g., Gairns 1953). Renewed attention to subtle morphological differences, characteristic of the earlier studies, will be needed to establish a hypothetical association between type of mechanoreceptive afferent (on the basis of response properties) and mode of termination in the tongue.

COMPARISON OF RECEPTIVE FIELD PROPERTIES WITH THOSE OF MECHANORECEPTORS IN OTHER PERIPHERAL NERVES IN HUMANS. Unequivocally, the human tongue is exquisitely sensitive to tactile stimulation (Lass et al. 1972; Ringel and Ewanowski 1965; Van Boven and Johnson 1994; for review see Rath and Essick 1990). Because the density of afferent units and the properties of the receptive fields (size and shape) are critical factors for the resolving power of the peripheral tactile system, the high spatial acuity for the tip of the tongue can be explained by two observations in the present study. First, the fascicle fields always included the tip of the tongue. A high overlap of fascicle fields is generally accepted as indicating a particularly high innervation density (e.g., see Hagbath et al. 1970; Johansson et al. 1988b; Nordin and Thomander 1989). Consistent with this interpretation, Marlow et al. (1965) found the tip of the human tongue to be the most highly innervated lingual site. Second, most of the superficial units had very small and well-defined receptive fields. In fact, the receptive field sizes, as well as the detection thresholds, found for the superficial RA and SA irregular mechanoreceptors in the tongue were smaller than those that have been reported for any other body area studied (see Edin et al. 1995; Johansson and Vallbo 1983; Vallbo et al. 1979).

This conclusion is supported by Fig. 3, B and C, in which are compared the receptive field sizes and the force thresholds for afferent units innervating four different regions of the human body: the tongue (lingual nerve), the glabrous skin of the hand (median nerve), the hairy skin of the hand (radial nerve), and the hairy skin of the face and red zone of the lip (mental nerve). Data from the median, radial, and mental nerves were collected in a similar manner to that employed in the present study and published previously (Edin et al. 1995). Analysis of variance of the combined data set revealed that receptive field size for the lingual nerve, on average, is less than that for the mental and radial nerves (P < 0.05). Moreover, thresholds for lingual nerve mechanoreceptors are less than those for any of the other three nerves studied (P < 0.05). The extremely low thresholds of the RA units (cf. Fig. 3C) and the high vascularity of the tongue may explain why 2 of the 14 units responded reliably to the arterial pulse. The precise mechanisms underlying this interesting phenomenon, however, remain to be investigated.

COMPARISON WITH MECHANORECEPTORS STUDIED IN THE LINGUAL NERVES OF OTHER SPECIES. The first recordings from mechanoreceptors in the cat's tongue were reported by Zotterman (1936) and later in more detail by Hensel and Zotterman (1951) and Hellekant (1965). Fast-conducting mechanoreceptive fibers from which the "largest spikes" were evoked (and presumably analogous to those reported in this paper, e.g., see Fig. 4 of Hellekant 1965) were identified in each investigation but were not systematically studied. A more slowly conducting (Adelta ) group of afferents responded not only to innocuous mechanical stimulation, but to cooling and to alcohol applied to the receptive field.

Porter (1966) and Biedenbach and Chan (1971) identified RA and SA single-unit responses from the feline lingual nerve when the tongue was stimulated mechanically. As in the present study, most afferents were judged to terminate superficially in the tongue and to exhibit very small receptive fields (1-4 mm2, Biedenbach and Chan 1971), often on or near the tip of the tongue. Of 39 superficially located units, Porter (1966) found all to adapt rapidly to a maintained mechanical stimulus, whereas Biedenbach and Chan (1971) found 74% of 28 units in their sample to be RA. Unlike in the present study, 45% of these RA units responded only to mechanical stimuli that moved across the receptive field ("stroking movement"). Biedenbach and Chan also found that the SA units could be divided into three groups on the basis of the duration of the response to maintained stimulation (viz., many min, several s, or only ~1 s).

SENSITIVITY TO TONGUE MOVEMENTS. In the present study, with one exception, the superficial units tested failed to respond to tongue movements in the absence of contact with other structures. Because the movements were relatively slow (0.5-1 Hz) in comparison with physiological movements (e.g., 6-7 Hz for speech) (Kuehn and Moll 1976), we cannot completely rule out the possibility that these afferents are stimulated during normal function in the absence of contact. However, this possibility seems unlikely on the basis of observations of the motion sensitivity of cutaneous mechanoreceptors supplying other body regions. For example, Johansson et al. (1988a) demonstrated that both SA and RA mechanoreceptors supplying the skin of the midface respond in a phase-dependent manner to rhythmic jaw ("chewing") movements with amplitudes and rates similar to those employed in the present experiments (~1 cm of maximum displacement and 1 Hz, respectively). More recently, Edin and Abbs (1991) demonstrated that SA I, SA II, and FA I mechanoreceptive afferents in the radial nerve respond to physiological movements of the digits: alternating flexion-extension movements about the metacarpophalangeal joint with frequencies of 0.5-1.5 Hz were highly effective in evoking discharge in these afferents. Moreover, the FA I units were found to be insensitive to mechanical transients generated by purposeful digit-to-digit contacts occurring at a distance from the receptive fields. On the basis of the pronounced motion sensitivity of cutaneous mechanoreceptors supplying the face and hand to relatively slow rates of movement, we hypothesize that faster movements of the tongue would not differ from slower movements in their inability to evoke discharge in the superficial mechanoreceptive afferents of the tongue.

Deep tongue mechanoreceptors

A minority of afferents examined in the present study responded to forceful probing of a large area of the tongue, indicating that they were situated deeply in the tongue muscle. These units all showed SA responses to forces applied perpendicularly to their receptive fields and were typically spontaneously active. Moreover, distinct differences in dynamic sensitivity to perpendicular indentation of the mucosa suggested that these units may have at least two different types of endorgans. The most likely candidates are the primary and secondary muscle spindle endings (B. B. Edin, personal communication), but Ruffini endings and Golgi tendon organs cannot be excluded. In addition to muscle spindles, these two endorgans have been identified in histological sections of primate tongue muscle (Bowman 1968; Cooper 1953; Fitzgerald and Sachithanandan 1979; Kubota et al. 1975).

COMPARISON WITH MECHANORECEPTORS STUDIED IN THE LINGUAL NERVES OF OTHER SPECIES. Mechanoreceptive afferents with characteristics similar to those of the deep units of the present study were described by Porter (1966) and Biedenbach and Chan (1971). Porter identified 23 afferents that responded to deep pressure and, to a lesser extent, to stretch applied to the tongue. Many of the units were spontaneously active and none responded to light mechanical stimulation of the mucosal surfaces of the tongue. Higher rates of discharge were evoked by greater degrees of deep deformation. Similarly, Biedenbach and Chan (1971) identified two afferents that responded only to "strong rolling movement of the probe over the tongue" (p. 587), which led these investigators to conclude that the units terminated deeply in the tongue musculature. These findings suggest that the lingual nerve of both cat and human provides a dual mechanoreceptive innervation (superficial and deep) and that many, but not all, characteristics of the innervations are common to both species.

SENSITIVITY TO TONGUE MOVEMENTS. In the present study all deep units tested responded vigorously in a phase-dependent manner to tongue movements. This suggests that the adequate stimulus for these units was tongue movement and/or muscle activity rather than indentation of the mucosa. Such units cannot be appropriately classified by their responses to perpendicular indentations of the mucosa, but rather should be classified by their responses to muscle activation and muscle stretch (see Edin and Vallbo 1990). However, no attempt was made during the recording sessions to further classify these afferents, because the ability to deliver controlled degrees of stretch to the lingual muscle of mechanoreceptor origin did not exist. Moreover, interpretation of responses to electrical muscle contraction is problematic (cf. Porter 1966).

Lingual proprioception and kinesthesis

It is generally accepted that the execution of purposeful and precise motor acts that characterize eating, drinking, and speaking require information about tongue position and movements. Human subjects also exhibit remarkably accurate and reliable performance on experimental tasks that necessarily require proprioceptive information. These tasks include 1) reproduction and bisection of sampled intervals of tongue protrusion (Porter and Lubker 1980), 2) scaling of distances between raised points on a plastic sheet covering the palate (Siegel and Hanlon 1983), 3) protrusion of the tongue in a specified direction (Grover and Craske 1991), and 4) rotation of the tongue to match the orientation shown in pictures (Benedetti 1988). The origin of the relevant sensory signals that contribute to motor control and perception, however, has been a matter of controversy over the years (e.g., Adatia and Gehring 1971; Benedetti 1988; Carleton 1938; Grover and Craske 1991; McDonald and Aungst 1970; Siegel and Hanlon 1983; Weddell et al. 1940).

The present study demonstrates that within the lingual nerve of humans are found deep mechanoreceptors that possess the capacity to signal tongue movements in the absence of direct contact with the receptive field, and that merit the label "proprioceptor" in a classical sense. The superficial mechanoreceptors do not possess this capacity, at least for relatively slow tongue movements. However, because the tongue is in close spatial proximity to stable landmarks (e.g., lower teeth and jaw), information about the location and movement of the tongue tip (i.e., proprioceptive information) is hypothetically available in the spatial and temporal patterns of contact-evoked discharge in the superficial mechanoreceptors supplying this region. Consistent with this hypothesis, Carleton (1938) demonstrated in an early study that after topical anesthesia of the lingual and oral mucosa (cocainization), only one of eight subjects could 100% correctly identify the direction to which the tongue was pulled by forceps. Carleton interpreted this to imply that conscious tongue position sense (kinesthesis) was normally mediated by spatial cues provided by contact of the lingual mucosa with stable intraoral landmarks.

The findings of Carleton (1938), however, have not been reproduced by other investigators. Weddell and colleagues (1940) anesthetized the sensory innervation of the tongue and lower face/jaw (viz., the lingual and inferior alveolar nerves) of 19 human subjects. Presumably this mode of anesthesia, unlike that employed by Carleton, blocked both the superficial and deep mechanoreceptors of the tongue, but subjects exhibited no ataxia or tendency to bite the tongue. Some difficulty in articulation was subjectively noted, however. Adatia and Gehring (1971) repeated the procedure on 12 subjects and found similarly that lingual kinesthesis was retained. The subjects could move the tongue in an instructed direction and report the direction to which the passive tongue was moved by the experimenters. Indeed, most studies have concluded that tongue position sense is mediated by deep mechanoreceptors unrelated to the lingual nerve. Moreover, these proprioceptors were hypothesized to travel in the hypoglossal nerve (Adatia and Gehring 1971; Cooper 1953; see also Bowman 1968; Fitzgerald and Sachithanandan 1979; Kubota et al. 1988).

That the lingual nerve does contribute to the motor control of the tongue is evidenced by objective studies reported in the speech and hearing literature: local anesthesia of the nerve results in well-documented errors in articulation (e.g., Ringel and Steer 1963). Moreover, cineradiographic studies of the tongue under lingual nerve block conditions indicate a decrease in the precision at which the tongue contacts other intraoral structures during articulation and a generalized retroflexion of the tip of the tongue (Putnam and Ringel 1976). A significant role for the superficial mechanoreceptors, at least in the absence of input from the deep mechanoreceptors, seems doubtful: topical anesthesia of the intraoral mucosa alone does not significantly alter speech production (Ringel and Steer 1963). Moreover, subjects' skills on tactile sensory tests (e.g., 2-point discrimination, stereognosis, or recognition of forms) that presumably utilize the superficial lingual innervation do not correlate with speech skills, suggesting a dissociation of the sensory signals contributing to the two capacities (McDonald and Aungst 1970).

More recent studies directly and convincingly demonstrate that lingual kinesthesis is mediated by deep receptors and that the superficial receptors play an apparently insignificant role (Benedetti 1988; Grover and Craske 1991; Siegel and Hanlon 1983). Grover and Craske (1991) studied subjects' percept of the straight-ahead position of the tongue before and after a task during which loads that attempted to pull the protruded tongue either rightward or leftward were resisted. Before loading, subjects exhibited an exceptional capacity to position the tongue as instructed. However, after resisting the horizontal load (after muscular effort), subjects protruded the tongue toward the side of the previous effort, suggesting that the tongue's perceived location had shifted away from that side. The error did not increase after topical anesthesia of the mucosa, suggesting that 1) the postcontraction bias was likely mediated by signals from deep muscle mechanoreceptors and 2) positional information provided by contact of the mucosa with familiar intraoral structures did not serve to minimize the bias in position sense.

Conversely, it has been shown that proprioceptive signals from the tongue muscles do not serve to reorient the mucosa's spatial frame of reference in response to physiological perturbations (Benedetti 1988). Under resting conditions, the tongue lies horizontally between the two halves of the bony mandible. Benedetti demonstrated that the spatial frame of reference for the mucosa remains in this position and is invariant with both actual and perceived changes in tongue position. Specifically, movement of a stimulus rod along the edge of the tongue tip was perceived as horizontally oriented (i.e., perceived as if the tongue remained in its resting position) even after subjects rotated the tongue tip 90°. As summarized by Benedetti: "whereas there is a system for changing the intrinsic coordinates of the tongue, there is no system for changing the spatial coordinates of tactile information" (p. 73). One implication is that an elongated food particle lying across and adhering to the tongue tip would not appear to change from a horizontal orientation during physiological rotations of the tongue. In contrast, the perceived orientation in space of stable intraoral landmarks, such as the lower teeth, would shift during function.

From the studies described above, one is led to the conclusion that central neural mechanisms underlying perception process information from the superficial mechanoreceptors and from the deep mechanoreceptors independently. Given the tremendous coordination of intraoral structures and actions required for function, the rationale for this perceptual independence is unclear and somewhat surprising. It is anticipated that basic knowledge of the mechanoreceptive innervation of the human tongue, such as provided by the microneurographic technique, will lead to the development of novel hypotheses and experiments to further clarify the roles of the two sources of sensory information in perceptual as well as in motor control functions.

    ACKNOWLEDGEMENTS

  This study was supported by Swedish Medical Research Council Grants 8667 and 11087 and National Institute of Dental Research Grants DE-10141 and DE-07509.

    FOOTNOTES

  Address for reprint requests: M. Trulsson, Dept. of Physiology, Umeå University, S-901 87 Umeå, Sweden.

  Received 3 July 1996; accepted in final form 3 October 1996.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society




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