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Trigeminal modulation of gustatory neurons in the nucleus of the solitary tract
Brain Research 973 (2003) 265–274 www.elsevier.com / locate / brainres
Research report
Trigeminal modulation of gustatory neurons in the nucleus of the solitary tract ´ a , E. Carstens b , * Yves Boucher a , Christopher T. Simons b , Annick Faurion c , Jean Azerad a
b
` , 75006 Paris, France UFR d’ Odontologie, 5 Rue Garanciere Section of Neurobiology, Physiology and Behavior, University of California, 1 Shields Avenue, Davis, CA 95616, USA c Laboratoire de Neurobiologie Sensorielle, 1 avenue des Olympiades, 91300 Massy, France Accepted 25 February 2003
Abstract Electrophysiological methods were used to investigate the effects of trigeminal nerve stimulation or transection on responses of single gustatory neurons in the nucleus of the solitary tract (NTS) to tastants (NaCl, sucrose, citric acid, monosodium glutamate) in pentobarbital-anesthetized rats. Unilateral transection of the lingual nerve, or the mandibular branch of the trigeminal nerve, resulted in significant reductions (by 21 and 29%, respectively; P,0.01) in tastant-evoked responses, with no further effect following bilateral transection. Electrical stimulation of the central cut end of the mandibular nerve directly excited nine of 14 gustatory NTS units. For these units, central mandibular stimulation facilitated the tastant-evoked responses in six, depressed responses in three, and had no effect in five. Facilitation of tastant-evoked responses peaked 4 min after mandibular stimulation and recovered within 8 min. Electrical stimulation of the peripheral cut end of the mandibular nerve significantly reduced tastant-evoked responses in nine other NTS units, with a maximal reduction at 4 min post-stimulation followed by recovery. Stimulation of the superior cervical sympathetic ganglion did not affect NTS tastant-evoked responses. These results suggest the presence of complex central modulation of NTS neurons by trigeminal afferents, as well as a peripheral depressant effect on gustatory processing possibly mediated via neuropeptide release from trigeminal nerve endings in the tongue. 2003 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Gustatory senses: central pathways Keywords: Trigeminal; Taste; Nucleus of solitary tract; Capsaicin; Modulation
1. Introduction Taste is a complex sensation that is initiated when sapid chemicals depolarize taste receptor cells located within taste buds of the oral cavity. Gustatory information is conveyed to the brainstem via the facial (cranial nerve VII), glossopharyngeal (cranial nerve IX) and vagus (cranial nerve X) nerves which terminate in a topographically organized manner in the nucleus of the solitary tract (NTS). During food ingestion, taste and somatosensory information are closely associated, and there is growing evidence that gustatory and trigeminal pathways may *Corresponding author. Tel.: 11-530-752-6640; fax: 11-530-7525582. E-mail address: [email protected] (E. Carstens).
interact. Trigeminal deafferentation reduced ingestive behaviors elicited by preferred tastants in rats [3], and patients with trigeminal disorders exhibit increased taste thresholds [16]. Peripheral gustatory chorda tympani ‘taste’ fibers also respond to thermal and mechanical stimuli [32,55], and electrical stimulation of the lingual nerve decreased responses of chorda tympani fibers to NaCl [51]. Centrally, somatosensory and gustatory inputs converge in the cortex [8,20], thalamus [38,40] and parabrachial nucleus [39], and anatomical studies in several species have revealed trigeminal projections to the rostro-lateral aspect of the NTS that receives heavy projections from the chorda tympani [1,2,19,26– 28,31,44,49,52,53]. Gustatory neurons in NTS often respond to somatosensory stimuli [38,50], and oral capsaicin (the pungent chemical in chili peppers) suppresses certain
0006-8993 / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02526-5
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taste qualities [45] as well as tastant-evoked responses of NTS neurons [46]. In the present study, we have used electrophysiological methods to further investigate trigeminal modulation of central gustatory processing. We tested if gustatory neurons in NTS show altered tastant-evoked responses following transection of the lingual or mandibular nerves, if electrical stimulation of the trigeminal ganglion directly affects NTS neurons, and if electrical stimulation of central or peripheral trigeminal fibers influences tastant-evoked responses of NTS neurons.
2. Materials and methods
2.1. Animals Twenty-eight male Sprague–Dawley rats (350–480 g; Simonsen, Gilroy, CA, USA) were used. They were housed two per cage in a vivarium maintained on a 12:12 h light:dark cycle at |21 8C. Food and water were available ad libitum. All procedures were in accordance with the NIH animal welfare guide and were approved by the University of California, Davis Animal Use and Care Advisory committee.
2.2. Surgery Animals were anesthetized with pentobarbital (65 mg / kg i.p.) and a midline incision was made over the trachea.
The hypoglossal nerve was cut bilaterally followed by tracheostomy and cannulation of the jugular vein to allow constant infusion of pentobarbital (10 mg kg 21 h 21 ). Anesthesia was monitored by absence of tachycardia (monitored by EKG) to noxious paw pinch. The head was fixed in a stereotaxic frame using atraumatic earbars, the cerebellum was aspirated to expose the medulla at the level of NTS, and a decerebration performed at the mid-collicular level to expose the trigeminal ganglia. The mouth was opened and the oral cavity was wetted with distilled water to prevent desiccation. Animals were ventilated with a rodent ventilator (Harvard Apparatus, model 683) at a rate and tidal volume that maintained end-tidal CO 2 (monitored with a Traverse 2000 capnometer) at 3–4%. Sites of nerve transection, stimulation, and NTS singleunit recording are shown schematically in Fig. 1. In six animals, the lingual nerve was exposed on each side by incision anterior to the ear. The temporal muscle was dissected to expose the mandibular condyle which was resected. The medial pterygoid muscle was carefully dissected to avoid bleeding of the adjacent venous sinuses and the lingual nerve was isolated after separation from the chorda tympani. A cotton thread was pulled loosely under the exposed nerve and saline-soaked cotton was placed gently in the wound to prevent dessication. Before transecting the lingual or mandibular (see below) nerve, the animals were paralyzed by infusion of pancuronium (0.2 mg / kg i.v.) in the femoral vein to prevent gross orofacial movements. After isolating and recording NTS single-unit tastant-evoked activity (see below), the ipsilateral lingual
Fig. 1. Schematic diagram showing sites of nerve transection or stimulation and NTS recording. The solid and dashed lines in trigeminal and gustatory primary afferent fibers, respectively. Thick dashed lines with numbers indicate sites of transection of contralateral (1b) lingual nerve and ipsilateral (2a) and contralateral (2b) mandibular nerve. Arrows with numbers indicate sites ipsilateral mandibular transection; 3 indicates the central cut end and 4 the peripheral cut end. NTS, nucleus of the solitary tract; V,
the tongue indicate ipsilateral (1a) and of stimulation after trigeminal complex.
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nerve was cut with ophthalmologic scissors (position 1a in Fig. 1) and removal of the intact thread was taken as evidence of complete transection. After recording the NTS response, the contralateral lingual nerve (position 1b in Fig. 1) was transected in the same manner. In 14 animals, the trigeminal ganglion was exposed bilaterally, the dura mater removed, and a cotton thread was pulled loosely underneath the mandibular division as it emerged proximally through the skull. The ipsilateral mandibular nerve (position 2a in Fig. 1) was cut distal to the ganglion with microscissors and removal of the intact thread was taken as evidence of complete transection. In six cases an NTS unit was isolated and its gustatory responses recorded before and after both ipsilateral (position 2a in Fig. 1) and contralateral (position 2b in Fig. 1) mandibular nerve transections. In these and the other eight cases in which NTS units were isolated after bilateral transactions, we also tested effects of electrical stimulation of the central or peripheral cut end of the mandibular nerve. In six control animals, the superior cervical sympathetic ganglion was exposed during the initial surgery, placed on a pair of silver wires, and isolated with dental silicon as described previously [23].
2.3. Recordings A teflon-insulated tungsten recording electrode (18–20 MV; F. Haer, Brunswick, ME, USA) was advanced into the brainstem (2.7 mm anterior to obex, 1.8 mm lateral to midline) using a hydraulic microdrive (David Kopf Instruments, Tujunga, CA, USA). Extracellular single-unit activity was amplified and displayed by conventional means and fed to a computer for analysis and storage [15] and displayed in peristimulus-time (PSTH, bin width: 1 s) format. A template-matching spike sorting procedure was used to discard artifacts [15]; examples of superimposed action potential waveforms from individual units are shown as insets in Figs. 2C, 3, 4C and 6B. Recordings were made from gustatory neurons in NTS prior to and 3 min following unilateral (ipsilateral) lingual or mandibular nerve transection, and again following transection on the contralateral side. Responses of some NTS units to mechanical (pressure applied by forceps) and thermal (hot water of |50 8C applied by syringe) stimulation of the tongue were also tested qualitatively. The effect of stimulating the central or peripheral cut end of the mandibular nerve on tastant-evoked responses was assessed immediately and 2, 4, 6 and 8 min post-stimulation. Single units responsive to gustatory stimuli were routinely observed at depths ranging from |700 to 1000 mm below the brain stem surface.
2.4. Chemical stimulation Gustatory NTS units were searched for using a taste mixture containing the following reagent grade chemicals:
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sucrose (0.3 M; Mallinkrodt, Paris, KY, USA), NaCl (0.1 M; Fisher Scientific, Fair Lawn, NJ, USA), citric acid (0.03 M; Mallinkrodt), and monosodium glutamate (0.2 M; Sigma, St. Louis, MO, USA) as in our previous study [46]. Units exhibiting an obvious increase in firing elicited by application of the taste mixture were tested further. Each of the four tastants was applied individually by syringe (|0.2 ml / s) to the anterior lingual surface for 15 s and left on for an additional 15 s, followed immediately by a distilled water rinse (3 ml). Activity was recorded beginning 30 s prior to the gustatory stimulus until 30 s after stimulus cessation. We considered the unit to respond if its firing rate increased by at least two- to threefold above its spontaneous firing rate. That this level of increase was sufficient is supported by post-hoc analysis showing that tastant-evoked responses were significantly greater than the spontaneous firing rate. Each tastant was applied at least twice to establish which elicited the relatively largest response. The most effective tastant identified in this manner was then reapplied three times successively to establish response reproducibility. Those units exhibiting stable tastant-evoked responses were then subjected to trigeminal manipulation and re-tested with the same tastant. All solutions were delivered at room temperature. Tastant-evoked responses were quantified as the total number of impulses during the 30-s stimulus period.
2.5. Electrical stimulation This was done following bilateral mandibular nerve transection. For stimulation of the central cut end of the ipsilateral mandibular nerve, a bipolar tungsten electrode connected to a stimulator (Grass S-88, Warwick, RI, USA) was inserted into the proximal cut end (position 3 in Fig. 1). A 20-s train of square-wave pulses (0.5 ms duration; 10 V) was delivered at 50 Hz. Previous studies used similar parameters to stimulate the trigeminal ganglion [13] or the lingual nerve [51]. Stimulus efficacy was verified by evoked jaw movement (in the unparalyzed condition). For stimulation of the peripheral cut end of the ipsilateral mandibular nerve, an electrode was inserted into the distal cut end (position 4 in Fig. 1). Identical parameters were used for stimulation of the superior cervical sympathetic ganglion (ipsilateral to recorded NTS unit) in separate animals.
2.6. Histology At the conclusion of each experiment an electrolytic lesion was made at the recording site by passing direct current (6 V) through the microelectrode for 1 min. Animals were killed by an overdose of pentobarbital delivered through the jugular cannula. The brainstems were removed and post-fixed in 10% formalin. At least 2 weeks later, they were cut in 50-mm coronal sections, counterstained with neutral red, and lesions were identified under the light microscope (Nikon E-400). All histologi-
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cally verified recording sites were at the rostral lateral border of the gustatory NTS [42].
2.7. Data analysis A direct excitatory or inhibitory effect of electrical stimulation of the central or peripheral end of the trigeminal ganglion, or superior cervical sympathetic ganglion, was taken as an increase or decrease, respectively, of .100% over the baseline spontaneous firing rate. The effect of electrical stimulation on tastant-evoked activity was assessed by first categorizing each unit according to whether its tastant-evoked response was increased, decreased, or not affected. The criterion for categorizing the unit’s response as increasing or decreasing was whether the change in size of the tastant-evoked response (single trial following nerve stimulation) exceeded 61.96 standard deviations of the mean of three tastant-evoked responses prior to electrical stimulation. The rationale is that a change in response of .1.96 S.D. falls outside of the 95% confidence interval. Averaged tastant-evoked responses prior to stimulation were then compared to the tastantevoked response elicited immediately, 2, 4, 6 and 8 min following stimulation using two-way analysis of variance (ANOVA; neuron and time as main effects) followed by post-hoc least significant difference (LSD) multiple comparison tests. Averaged tastant-evoked responses were similarly compared pre- and post-transection of the lingual or mandibular nerve. A P,0.05 was taken to be significant.
3. Results
3.1. Reduced tastant-evoked responses following lingual and mandibular nerve transection Ipsilateral transection of the lingual nerve resulted in a significant (P50.013) 21% decrease in the tastant-evoked responses of six NTS units (three citric acid, two sucrose, one NaCl). Fig. 2A plots each unit’s tastant-evoked response prior to nerve transection against its response following transection (open squares), with the dashed diagonal indicating no effect. All data points lie beneath the diagonal, indicating that responses of all units were reduced to variable degrees. The open bars in Fig. 2B show that mean responses were significantly lower following transection of the ipsilateral lingual nerve with no further effect following transection of the contralateral lingual nerve. Fig. 2C shows an individual example of an NTS unit in which sucrose-evoked responses were reduced following ipsilateral lingual nerve transection. Similarly, ipsilateral mandibular nerve transection reduced the tastant-evoked (three citric acid, one sucrose, one NaCl, one MSG) responses of six other NTS units to variable degrees (filled circles in Fig. 2A). The mean
reduction by 29% was significant (P50.007; Fig. 2B, filled bars) and was not further affected following transection of the contralateral mandibular nerve. Transection of neither the lingual nor the mandibular nerve had any significant effect on the mean level of spontaneous firing measured during the 30-s period prior to the application of tastants (lingual nerve: 3.661.3 (S.D.) impulses / s pre, 3.362.4 after ipsilateral; 3.362.8 after bilateral transection; mandibular nerve: 2.661.3 impulses / s pre, 1.961.3 after ipsilateral, 2.361.5 after bilateral transection). Following bilateral mandibular nerve transection, five gustatory NTS units tested also responded to somatosensory (tactile and warming) stimulation of the tongue, consistent with the possibility that they received input from chorda tympani fibers with somatosensory properties [32,55].
3.2. Direct excitation of NTS units by central mandibular stimulation Following mandibular nerve transection, the effect of electrical stimulation of the central cut end of the ipsilateral mandibular nerve was tested in 14 NTS units; nine exhibited increased firing while the remaining five were unaffected. In one case, direct excitation was followed by reduced firing as illustrated in Fig. 3.
3.3. Effects of central mandibular stimulation on NTS tastant-evoked responses Effects of central mandibular stimulation on tastantevoked responses were tested in the same 14 NTS units. Fig. 4A is a scatter plot in the same format as Fig. 2A, plotting each unit’s tastant-evoked response before vs. the response immediately after electrical stimulation of the central cut end of the mandibular nerve. Responses of six units were significantly (P50.019) facilitated (.1.96 S.D. above mean baseline level; open symbols in Fig. 4A), while responses of three were reduced (shaded symbols in Fig. 4A) and responses of five units were unchanged (filled symbols in Fig. 4A). Responses of the six units exhibiting facilitation are plotted vs. time in Fig. 4B to show that the increase became significant within 2 min following electrical stimulation with recovery by 8 min. An individual example of facilitation of sucrose-evoked responses following central mandibular stimulation is shown in Fig. 4C. For the three units exhibiting reduced responses, the mean change was not statistically significant (P50.134). The effect of central mandibular stimulation on tastantevoked responses did not appear to correlate with the presence or absence of direct excitation. Of the nine units driven directly by central mandibular stimulation, tastantevoked responses were increased in four, decreased in three and unaffected in two. One of these was a unit that was initially excited directly by central mandibular stimu-
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Fig. 2. Reduction in tastant-evoked responses following lingual or mandibular nerve transection. (A) Scatter plot graphing each neuron’s response to the tastant (mean of three trials) prior to trigeminal stimulation (horizontal axis) vs. its response to the same tastant (single trial) following electrical stimulation of the central cut end of the mandibular nerve (vertical axis). Dashed diagonal indicates line of equality (i.e. no effect of trigeminal stimulation). Open squares, ipsilateral lingual nerve transection; filled circles, ipsilateral mandibular nerve transection. (B) Bar graph plots mean tastant-evoked response of NTS neurons prior to (pre, left-hand bars) and following ipsilateral (ipsi, middle bars) or bilateral (bilat, right-hand bars) transection of the lingual nerve (open bars) or mandibular branch of the trigeminal nerve (filled bars). Error bars in this and subsequent figures: S.E.M. *Significantly different from pre (ANOVA, P,0.05). (C) Individual example. Shown are peristimulus-time histograms (PSTHs; bin width, 1 s) of a gustatory NTS unit’s responses to lingual application of 0.3 M sucrose (upward and downward arrows in upper bars indicate onset and cessation of stimulus) before (left-hand PSTH) and after ipsilateral (middle PSTH) and bilateral (right-hand PSTH) transection of the lingual nerve. Upper right-hand inset (in box) shows a series of 15 successively recorded action potential waveforms superimposed.
lation followed by reduced firing. Interestingly, its tastantevoked response exhibited a similar biphasic pattern of initial facilitation followed by depression (Fig. 3, righthand PSTH). Of the five NTS units not directly driven by central mandibular stimulation, tastant-evoked responses were increased in two and unaffected in three.
3.4. Depression of tastant-evoked NTS responses by peripheral mandibular nerve stimulation In eight NTS units (four citric acid, three sucrose, one NaCl), electrical stimulation of the peripheral trunk of the sectioned ipsilateral mandibular nerve consistently reduced
tastant-evoked responses to variable degrees, with an overall significant decrease (P50.022) that reached a nadir at 2–4 min post-stimulation and returned to pre-stimulation levels thereafter (Fig. 5).
3.5. Sympathetic stimulation Stimulation of the ipsilateral superior cervical sympathetic ganglion did not result in any significant (P50.798) change in tastant-evoked responses of six NTS units tested (two citric acid, three sucrose, one NaCl). Fig. 6A shows that the mean tastant-evoked responses of these units were unaffected following ganglionic stimulation (applied prior
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Fig. 3. Example of gustatory NTS unit driven by electrical stimulation of the central cut end of the mandibular nerve. PSTHs show, from left to right, the unit’s response to citric acid (upper bar with arrows), trigeminal stimulation alone (TG stim., at middle bar), and trigeminal stimulation (at right-hand bar) followed immediately afterward by citric acid. Note that TG stimulation excited the unit followed by inhibition (middle PSTH), and similarly facilitated and then inhibited the response to citric acid (right-hand PSTH). Upper inset shows superimposed action potential waveforms.
to trial 2). Since ganglionic stimulation had no effect, these data further illustrate the relative stability of tastant-evoked responses across repeated trials. Fig. 6B shows an individual example of the stable responses of a unit to repeated applications of citric acid.
4. Discussion The present study provides evidence for direct and / or indirect modulatory effects of the trigeminal nerve on gustatory processing by NTS neurons. Unilateral transection of the lingual or mandibular nerve resulted in a 20–30% decrease in the magnitude of tastant-evoked responses of NTS neurons. Furthermore, electrical stimulation of the central end of the sectioned mandibular nerve directly excited nine of 14 gustatory NTS neurons, consistent with the possibility that some trigeminal primary afferents may have direct excitatory projections to NTS. Central mandibular stimulation variably affected tastantevoked responses of NTS cells, with 43% showing an increase and 22% a decrease. Finally, electrical stimulation of the peripheral end of the sectioned mandibular nerve resulted in an |20% decrease in the tastant-evoked responses of gustatory NTS neurons. These findings are discussed in terms of possible underlying neural mechanisms and behavioral correlates.
Fig. 4. Effects of central trigeminal stimulation on tastant-evoked responses of NTS neurons. (A) Scatter plot as in Fig. 2A graphing each neuron’s response to the tastant before and after stimulation of the central cut end of the mandibular nerve. Filled symbols, response not affected; open symbols, response increased; shaded symbols, response reduced. (B) Bar graph plots mean responses vs. time for six units whose responses increased (by .1.96 S.D. of mean baseline level) after trigeminal nerve stimulation. Error bars, S.E.M. **Significantly different (ANOVA, P, 0.05) from control response (pre). (C) Individual example of NTS unit’s successive responses to NaCl relative to an episode of central trigeminal nerve stimulation (indicated by bar). Note the transient increase in NaCl-evoked response size following TG stimulation (2nd–4th PSTHs from left). Format as in Fig. 2C.
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Fig. 5. Depression of NTS tastant-evoked responses by electrical stimulation of the peripheral cut end of the mandibular nerve. Graph as in Fig. 4B, plotting mean tastant-evoked responses over time. *Significantly different from pre (P,0.05).
4.1. Reduced gustatory NTS neuronal responses after trigeminal nerve transection The presently observed reduction in the magnitude of NTS neuronal tastant-evoked responses following transection of the lingual or mandibular nerves might be explained by (a) non-specific consequences of nerve transection (e.g. injury discharge) which might produce acute peripheral changes that altered the chorda tympani’s sensitivity to tastants, as discussed later, (b) the elimination of trigeminal input evoked by tastant stimuli, or (c) removal of tonic trigeminal excitation of NTS (disfacilitation). Regarding the second possibility, citric acid at high (250–300 mM) concentrations excites lingual nerve [6] and trigeminal subnucleus caudalis (Vc) neurons [48] and induces oral irritation [12]; the lower concentration used presently (30 mM), while not irritating, may conceivably have activated lingual fibers [43]. Therefore, eliminating input from citric acid-sensitive trigeminal fibers that project into NTS might partly account for the reduced responsiveness of these neurons to this stimulus post-nerve transection. However, this explanation is less likely for NaCl which excites the lingual nerve [47] and central Vc neurons [7] at higher concentrations than used presently, as well as for sucrose which is generally considered not to excite trigeminal afferents. Another possibility is that trigeminal nerve transection removed a tonic excitation of NTS neurons, rendering them less excitable and hence reducing their tastant-evoked responses. Electrical stimulation of the central trunk of the sectioned mandibular nerve predominantly excited gustatory NTS neurons. If mandibular afferent fibers having
Fig. 6. Absence of effect of stimulation of superior cervical sympathetic ganglion on NTS unit tastant-evoked responses. (A) Bar graph plotting mean tastant-evoked responses of six units across trials of repeated tastant stimulation. The superior cervical sympathetic ganglion was electrically stimulated prior to trial 2. (B) Individual example of an NTS unit’s stable responses to repeated application of citric acid. The superior cervical sympathetic ganglion was electrically stimulated prior to the second stimulus. Inset shows superimposed action potential waveforms.
excitatory synapses in NTS were tonically active, this could result in tonic depolarization of NTS neurons. Transection of the lingual or mandibular nerves would then remove this tonic facilitation to result in a reduction (disfacilitation) in tastant-evoked responsiveness of NTS neurons as was presently observed. This mechanism is consistent with the clinical observation that patients with trigeminal disorders possibly associated with partial deafferentation exhibit higher taste detection thresholds as assessed by electrogustometry [4,16]. That transection of the lingual nerve innervating the tongue had a somewhat smaller depressant effect on tastant-evoked responses compared to mandibular transection (Fig. 2A) suggests that any tonic trigeminal facilitatory effect originates not just from lingual afferents but also from mandibular afferents
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innervating other tissues as well. However, transection of neither the mandibular nor the lingual nerves had any significant effect on the spontaneous firing rate of gustatory NTS neurons, arguing against the notion that these nerves provide a tonic facilitatory input to NTS.
4.2. Modulation of gustatory NTS neurons by central mandibular stimulation Electrical stimulation of the central cut end of the mandibular nerve directly excited a majority (nine of 14) of gustatory NTS units and variably affected their tastantevoked responses, with six facilitated, three reduced and five unaffected. These effects might be mediated via direct primary afferent input to NTS (see Introduction), as well as indirect projections to NTS from second- or higher-order neurons in brain stem trigeminal subnuclei [34]. Some trigeminal primary afferents release substance P or other neuropeptides from their central terminals [9,13]. Neurokinin 1 receptors, to which substance P preferentially binds, have been identified in the rostrolateral NTS [11] that receives trigeminal projections [19,26–28,31,49]. Microinjection of substance P into the NTS variably modulated responses of gustatory NTS neurons, with 48% showing an increase and 9% a decrease in firing [10] similar to the percentages of NTS neurons exhibiting increases and decreases in tastant-evoked responses following central mandibular stimulation observed here. We cannot completely rule out a possible parasympathetic effect on lingual blood flow which might indirectly affect NTS unit tastant-evoked responses when the central cut end of the mandibular nerve was stimulated. In the cat, electrical stimulation of the central cut end of the lingual nerve elicits increases in blood flow to oral structures [25] possibly via a parasympathetically-mediated vasodilation [24]. However, this should affect neuronal responses consistently, while we observed mixed effects following central mandibular stimulation. In this regard, a parasympathetically-mediated increase in salivary flow might indirectly affect tastant-evoked responses [17], but any increase in salivary volume would be small compared to the volume of tastant applied by constant flow and hence unlikely to appreciably change the tastant concentration.
4.3. Depression of gustatory NTS neurons by peripheral mandibular stimulation In contrast to the mixed but predominantly excitatory effect of central mandibular nerve stimulation, electrical stimulation of the distal cut end of the mandibular nerve had a predominantly depressant effect on gustatory responses of NTS neurons. It was shown previously that electrical stimulation of the lingual nerve [51], as well as intraoral capsaicin [41], suppressed NaCl-evoked responses in chorda tympani fibers. We have recently reported that lingual capsaicin also reduces the tastant-
evoked responses of NTS neurons largely through a peripheral mechanism, since capsaicin depression of tastant-evoked NTS responses was equally robust following complete bilateral trigeminal ganglionectomy [46]. One possibility is that trigeminal nerve stimulation evokes the release of substance P and / or other neuropeptides from the peripheral nerve endings in the tongue. Peripheral nerve fibers of trigeminal origin are found in high density in and around taste buds [14,35,37,54]. It was proposed that the local release of neuropeptides from trigeminal nerve endings might decrease the sensitivity of taste receptor cells, explaining capsaicin and lingual nerve stimulationevoked reductions in responses of chorda tympani fibers to NaCl [41,46,51]. However, given that sympathetic nerve fibers are present in the trigeminal ganglion [33], it is also possible that activation of sympathetic reflexes (e.g. blood flow changes) might contribute to the suppression of tastant-evoked NTS responses following electrical nerve stimulation. Prior studies have shown that altering blood pressure can modulate the magnitude of tastant-evoked responses in chorda tympani fibers [21,22]. An indirect sympathetically-mediated effect is mitigated by our present finding that electrical stimulation of the superior cervical sympathetic ganglion did not significantly alter tastantevoked responses of NTS neurons. It is interesting to note that in our previous study, lingual capsaicin application suppressed NTS taste responses to about 60% of pre-capsaicin levels [46], an effect that was greater than that of peripheral mandibular nerve stimulation (to about 80% of control). This suggests that capsaicin is more effective than electrical nerve stimulation to evoke peripheral neuropeptide release and / or to engage other mechanisms to suppress gustatory transmission [46]. It is conceivable that local alterations in lingual temperature may have contributed to the observed effects. Tastantevoked responses are thermally sensitive [36] and a prior study, using the same stimulation parameters used here, noted an |5 8C increase in rat tongue temperature [51]. However, such a thermal effect would be reduced to some extent in our study, since tastants were delivered by constant flow at room temperature such that the fluid volume on the tongue would ‘clamp’ the tongue temperature at a fairly constant level.
4.4. Behavioral correlates Interactions between cranial nerves innervating the tongue have been documented before [5], and several groups have proposed that the chorda tympani normally inhibits activity in the glossopharyngeal nerve [18,29]. This interaction is presumed to preserve taste function when damage to the chorda tympani and / or anterior portion of the tongue occurs [18,29]. Taste and somatosensory stimuli are invariably linked during mastication and swallowing. Because taste conveys invaluable information regarding the acceptability of foods, it seems
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reasonable that somatosensory information carried by trigeminal afferents into the NTS would modulate gustatory activity to make it more physiologically meaningful. For instance, it has been reported that the trigeminal nerve is sensitive to quinine [30,43], and one may speculate that enhanced trigeminal activity could potentiate NTS gustatory responses to quinine. Further studies are needed to elucidate the behavioral significance of the modulatory effects of the trigeminal nerve on gustatory processing described here.
[12]
[13]
[14]
[15]
Acknowledgements This work was supported by grants from the International Association for the Study of Pain, the Institut Francais pour la Recherche Odontologique, the California Tobaccorelated Disease Research Program ([ 10DT-0197 and 11RT-0053), and the National Institute of Dental and Craniofacial Research ([ DR13685). The authors gratefully acknowledge the excellent histological assistance of M. Iodi Carstens.
[16]
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Research report
Trigeminal modulation of gustatory neurons in the nucleus of the solitary tract ´ a , E. Carstens b , * Yves Boucher a , Christopher T. Simons b , Annick Faurion c , Jean Azerad a
b
` , 75006 Paris, France UFR d’ Odontologie, 5 Rue Garanciere Section of Neurobiology, Physiology and Behavior, University of California, 1 Shields Avenue, Davis, CA 95616, USA c Laboratoire de Neurobiologie Sensorielle, 1 avenue des Olympiades, 91300 Massy, France Accepted 25 February 2003
Abstract Electrophysiological methods were used to investigate the effects of trigeminal nerve stimulation or transection on responses of single gustatory neurons in the nucleus of the solitary tract (NTS) to tastants (NaCl, sucrose, citric acid, monosodium glutamate) in pentobarbital-anesthetized rats. Unilateral transection of the lingual nerve, or the mandibular branch of the trigeminal nerve, resulted in significant reductions (by 21 and 29%, respectively; P,0.01) in tastant-evoked responses, with no further effect following bilateral transection. Electrical stimulation of the central cut end of the mandibular nerve directly excited nine of 14 gustatory NTS units. For these units, central mandibular stimulation facilitated the tastant-evoked responses in six, depressed responses in three, and had no effect in five. Facilitation of tastant-evoked responses peaked 4 min after mandibular stimulation and recovered within 8 min. Electrical stimulation of the peripheral cut end of the mandibular nerve significantly reduced tastant-evoked responses in nine other NTS units, with a maximal reduction at 4 min post-stimulation followed by recovery. Stimulation of the superior cervical sympathetic ganglion did not affect NTS tastant-evoked responses. These results suggest the presence of complex central modulation of NTS neurons by trigeminal afferents, as well as a peripheral depressant effect on gustatory processing possibly mediated via neuropeptide release from trigeminal nerve endings in the tongue. 2003 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Gustatory senses: central pathways Keywords: Trigeminal; Taste; Nucleus of solitary tract; Capsaicin; Modulation
1. Introduction Taste is a complex sensation that is initiated when sapid chemicals depolarize taste receptor cells located within taste buds of the oral cavity. Gustatory information is conveyed to the brainstem via the facial (cranial nerve VII), glossopharyngeal (cranial nerve IX) and vagus (cranial nerve X) nerves which terminate in a topographically organized manner in the nucleus of the solitary tract (NTS). During food ingestion, taste and somatosensory information are closely associated, and there is growing evidence that gustatory and trigeminal pathways may *Corresponding author. Tel.: 11-530-752-6640; fax: 11-530-7525582. E-mail address: [email protected] (E. Carstens).
interact. Trigeminal deafferentation reduced ingestive behaviors elicited by preferred tastants in rats [3], and patients with trigeminal disorders exhibit increased taste thresholds [16]. Peripheral gustatory chorda tympani ‘taste’ fibers also respond to thermal and mechanical stimuli [32,55], and electrical stimulation of the lingual nerve decreased responses of chorda tympani fibers to NaCl [51]. Centrally, somatosensory and gustatory inputs converge in the cortex [8,20], thalamus [38,40] and parabrachial nucleus [39], and anatomical studies in several species have revealed trigeminal projections to the rostro-lateral aspect of the NTS that receives heavy projections from the chorda tympani [1,2,19,26– 28,31,44,49,52,53]. Gustatory neurons in NTS often respond to somatosensory stimuli [38,50], and oral capsaicin (the pungent chemical in chili peppers) suppresses certain
0006-8993 / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02526-5
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taste qualities [45] as well as tastant-evoked responses of NTS neurons [46]. In the present study, we have used electrophysiological methods to further investigate trigeminal modulation of central gustatory processing. We tested if gustatory neurons in NTS show altered tastant-evoked responses following transection of the lingual or mandibular nerves, if electrical stimulation of the trigeminal ganglion directly affects NTS neurons, and if electrical stimulation of central or peripheral trigeminal fibers influences tastant-evoked responses of NTS neurons.
2. Materials and methods
2.1. Animals Twenty-eight male Sprague–Dawley rats (350–480 g; Simonsen, Gilroy, CA, USA) were used. They were housed two per cage in a vivarium maintained on a 12:12 h light:dark cycle at |21 8C. Food and water were available ad libitum. All procedures were in accordance with the NIH animal welfare guide and were approved by the University of California, Davis Animal Use and Care Advisory committee.
2.2. Surgery Animals were anesthetized with pentobarbital (65 mg / kg i.p.) and a midline incision was made over the trachea.
The hypoglossal nerve was cut bilaterally followed by tracheostomy and cannulation of the jugular vein to allow constant infusion of pentobarbital (10 mg kg 21 h 21 ). Anesthesia was monitored by absence of tachycardia (monitored by EKG) to noxious paw pinch. The head was fixed in a stereotaxic frame using atraumatic earbars, the cerebellum was aspirated to expose the medulla at the level of NTS, and a decerebration performed at the mid-collicular level to expose the trigeminal ganglia. The mouth was opened and the oral cavity was wetted with distilled water to prevent desiccation. Animals were ventilated with a rodent ventilator (Harvard Apparatus, model 683) at a rate and tidal volume that maintained end-tidal CO 2 (monitored with a Traverse 2000 capnometer) at 3–4%. Sites of nerve transection, stimulation, and NTS singleunit recording are shown schematically in Fig. 1. In six animals, the lingual nerve was exposed on each side by incision anterior to the ear. The temporal muscle was dissected to expose the mandibular condyle which was resected. The medial pterygoid muscle was carefully dissected to avoid bleeding of the adjacent venous sinuses and the lingual nerve was isolated after separation from the chorda tympani. A cotton thread was pulled loosely under the exposed nerve and saline-soaked cotton was placed gently in the wound to prevent dessication. Before transecting the lingual or mandibular (see below) nerve, the animals were paralyzed by infusion of pancuronium (0.2 mg / kg i.v.) in the femoral vein to prevent gross orofacial movements. After isolating and recording NTS single-unit tastant-evoked activity (see below), the ipsilateral lingual
Fig. 1. Schematic diagram showing sites of nerve transection or stimulation and NTS recording. The solid and dashed lines in trigeminal and gustatory primary afferent fibers, respectively. Thick dashed lines with numbers indicate sites of transection of contralateral (1b) lingual nerve and ipsilateral (2a) and contralateral (2b) mandibular nerve. Arrows with numbers indicate sites ipsilateral mandibular transection; 3 indicates the central cut end and 4 the peripheral cut end. NTS, nucleus of the solitary tract; V,
the tongue indicate ipsilateral (1a) and of stimulation after trigeminal complex.
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nerve was cut with ophthalmologic scissors (position 1a in Fig. 1) and removal of the intact thread was taken as evidence of complete transection. After recording the NTS response, the contralateral lingual nerve (position 1b in Fig. 1) was transected in the same manner. In 14 animals, the trigeminal ganglion was exposed bilaterally, the dura mater removed, and a cotton thread was pulled loosely underneath the mandibular division as it emerged proximally through the skull. The ipsilateral mandibular nerve (position 2a in Fig. 1) was cut distal to the ganglion with microscissors and removal of the intact thread was taken as evidence of complete transection. In six cases an NTS unit was isolated and its gustatory responses recorded before and after both ipsilateral (position 2a in Fig. 1) and contralateral (position 2b in Fig. 1) mandibular nerve transections. In these and the other eight cases in which NTS units were isolated after bilateral transactions, we also tested effects of electrical stimulation of the central or peripheral cut end of the mandibular nerve. In six control animals, the superior cervical sympathetic ganglion was exposed during the initial surgery, placed on a pair of silver wires, and isolated with dental silicon as described previously [23].
2.3. Recordings A teflon-insulated tungsten recording electrode (18–20 MV; F. Haer, Brunswick, ME, USA) was advanced into the brainstem (2.7 mm anterior to obex, 1.8 mm lateral to midline) using a hydraulic microdrive (David Kopf Instruments, Tujunga, CA, USA). Extracellular single-unit activity was amplified and displayed by conventional means and fed to a computer for analysis and storage [15] and displayed in peristimulus-time (PSTH, bin width: 1 s) format. A template-matching spike sorting procedure was used to discard artifacts [15]; examples of superimposed action potential waveforms from individual units are shown as insets in Figs. 2C, 3, 4C and 6B. Recordings were made from gustatory neurons in NTS prior to and 3 min following unilateral (ipsilateral) lingual or mandibular nerve transection, and again following transection on the contralateral side. Responses of some NTS units to mechanical (pressure applied by forceps) and thermal (hot water of |50 8C applied by syringe) stimulation of the tongue were also tested qualitatively. The effect of stimulating the central or peripheral cut end of the mandibular nerve on tastant-evoked responses was assessed immediately and 2, 4, 6 and 8 min post-stimulation. Single units responsive to gustatory stimuli were routinely observed at depths ranging from |700 to 1000 mm below the brain stem surface.
2.4. Chemical stimulation Gustatory NTS units were searched for using a taste mixture containing the following reagent grade chemicals:
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sucrose (0.3 M; Mallinkrodt, Paris, KY, USA), NaCl (0.1 M; Fisher Scientific, Fair Lawn, NJ, USA), citric acid (0.03 M; Mallinkrodt), and monosodium glutamate (0.2 M; Sigma, St. Louis, MO, USA) as in our previous study [46]. Units exhibiting an obvious increase in firing elicited by application of the taste mixture were tested further. Each of the four tastants was applied individually by syringe (|0.2 ml / s) to the anterior lingual surface for 15 s and left on for an additional 15 s, followed immediately by a distilled water rinse (3 ml). Activity was recorded beginning 30 s prior to the gustatory stimulus until 30 s after stimulus cessation. We considered the unit to respond if its firing rate increased by at least two- to threefold above its spontaneous firing rate. That this level of increase was sufficient is supported by post-hoc analysis showing that tastant-evoked responses were significantly greater than the spontaneous firing rate. Each tastant was applied at least twice to establish which elicited the relatively largest response. The most effective tastant identified in this manner was then reapplied three times successively to establish response reproducibility. Those units exhibiting stable tastant-evoked responses were then subjected to trigeminal manipulation and re-tested with the same tastant. All solutions were delivered at room temperature. Tastant-evoked responses were quantified as the total number of impulses during the 30-s stimulus period.
2.5. Electrical stimulation This was done following bilateral mandibular nerve transection. For stimulation of the central cut end of the ipsilateral mandibular nerve, a bipolar tungsten electrode connected to a stimulator (Grass S-88, Warwick, RI, USA) was inserted into the proximal cut end (position 3 in Fig. 1). A 20-s train of square-wave pulses (0.5 ms duration; 10 V) was delivered at 50 Hz. Previous studies used similar parameters to stimulate the trigeminal ganglion [13] or the lingual nerve [51]. Stimulus efficacy was verified by evoked jaw movement (in the unparalyzed condition). For stimulation of the peripheral cut end of the ipsilateral mandibular nerve, an electrode was inserted into the distal cut end (position 4 in Fig. 1). Identical parameters were used for stimulation of the superior cervical sympathetic ganglion (ipsilateral to recorded NTS unit) in separate animals.
2.6. Histology At the conclusion of each experiment an electrolytic lesion was made at the recording site by passing direct current (6 V) through the microelectrode for 1 min. Animals were killed by an overdose of pentobarbital delivered through the jugular cannula. The brainstems were removed and post-fixed in 10% formalin. At least 2 weeks later, they were cut in 50-mm coronal sections, counterstained with neutral red, and lesions were identified under the light microscope (Nikon E-400). All histologi-
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cally verified recording sites were at the rostral lateral border of the gustatory NTS [42].
2.7. Data analysis A direct excitatory or inhibitory effect of electrical stimulation of the central or peripheral end of the trigeminal ganglion, or superior cervical sympathetic ganglion, was taken as an increase or decrease, respectively, of .100% over the baseline spontaneous firing rate. The effect of electrical stimulation on tastant-evoked activity was assessed by first categorizing each unit according to whether its tastant-evoked response was increased, decreased, or not affected. The criterion for categorizing the unit’s response as increasing or decreasing was whether the change in size of the tastant-evoked response (single trial following nerve stimulation) exceeded 61.96 standard deviations of the mean of three tastant-evoked responses prior to electrical stimulation. The rationale is that a change in response of .1.96 S.D. falls outside of the 95% confidence interval. Averaged tastant-evoked responses prior to stimulation were then compared to the tastantevoked response elicited immediately, 2, 4, 6 and 8 min following stimulation using two-way analysis of variance (ANOVA; neuron and time as main effects) followed by post-hoc least significant difference (LSD) multiple comparison tests. Averaged tastant-evoked responses were similarly compared pre- and post-transection of the lingual or mandibular nerve. A P,0.05 was taken to be significant.
3. Results
3.1. Reduced tastant-evoked responses following lingual and mandibular nerve transection Ipsilateral transection of the lingual nerve resulted in a significant (P50.013) 21% decrease in the tastant-evoked responses of six NTS units (three citric acid, two sucrose, one NaCl). Fig. 2A plots each unit’s tastant-evoked response prior to nerve transection against its response following transection (open squares), with the dashed diagonal indicating no effect. All data points lie beneath the diagonal, indicating that responses of all units were reduced to variable degrees. The open bars in Fig. 2B show that mean responses were significantly lower following transection of the ipsilateral lingual nerve with no further effect following transection of the contralateral lingual nerve. Fig. 2C shows an individual example of an NTS unit in which sucrose-evoked responses were reduced following ipsilateral lingual nerve transection. Similarly, ipsilateral mandibular nerve transection reduced the tastant-evoked (three citric acid, one sucrose, one NaCl, one MSG) responses of six other NTS units to variable degrees (filled circles in Fig. 2A). The mean
reduction by 29% was significant (P50.007; Fig. 2B, filled bars) and was not further affected following transection of the contralateral mandibular nerve. Transection of neither the lingual nor the mandibular nerve had any significant effect on the mean level of spontaneous firing measured during the 30-s period prior to the application of tastants (lingual nerve: 3.661.3 (S.D.) impulses / s pre, 3.362.4 after ipsilateral; 3.362.8 after bilateral transection; mandibular nerve: 2.661.3 impulses / s pre, 1.961.3 after ipsilateral, 2.361.5 after bilateral transection). Following bilateral mandibular nerve transection, five gustatory NTS units tested also responded to somatosensory (tactile and warming) stimulation of the tongue, consistent with the possibility that they received input from chorda tympani fibers with somatosensory properties [32,55].
3.2. Direct excitation of NTS units by central mandibular stimulation Following mandibular nerve transection, the effect of electrical stimulation of the central cut end of the ipsilateral mandibular nerve was tested in 14 NTS units; nine exhibited increased firing while the remaining five were unaffected. In one case, direct excitation was followed by reduced firing as illustrated in Fig. 3.
3.3. Effects of central mandibular stimulation on NTS tastant-evoked responses Effects of central mandibular stimulation on tastantevoked responses were tested in the same 14 NTS units. Fig. 4A is a scatter plot in the same format as Fig. 2A, plotting each unit’s tastant-evoked response before vs. the response immediately after electrical stimulation of the central cut end of the mandibular nerve. Responses of six units were significantly (P50.019) facilitated (.1.96 S.D. above mean baseline level; open symbols in Fig. 4A), while responses of three were reduced (shaded symbols in Fig. 4A) and responses of five units were unchanged (filled symbols in Fig. 4A). Responses of the six units exhibiting facilitation are plotted vs. time in Fig. 4B to show that the increase became significant within 2 min following electrical stimulation with recovery by 8 min. An individual example of facilitation of sucrose-evoked responses following central mandibular stimulation is shown in Fig. 4C. For the three units exhibiting reduced responses, the mean change was not statistically significant (P50.134). The effect of central mandibular stimulation on tastantevoked responses did not appear to correlate with the presence or absence of direct excitation. Of the nine units driven directly by central mandibular stimulation, tastantevoked responses were increased in four, decreased in three and unaffected in two. One of these was a unit that was initially excited directly by central mandibular stimu-
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Fig. 2. Reduction in tastant-evoked responses following lingual or mandibular nerve transection. (A) Scatter plot graphing each neuron’s response to the tastant (mean of three trials) prior to trigeminal stimulation (horizontal axis) vs. its response to the same tastant (single trial) following electrical stimulation of the central cut end of the mandibular nerve (vertical axis). Dashed diagonal indicates line of equality (i.e. no effect of trigeminal stimulation). Open squares, ipsilateral lingual nerve transection; filled circles, ipsilateral mandibular nerve transection. (B) Bar graph plots mean tastant-evoked response of NTS neurons prior to (pre, left-hand bars) and following ipsilateral (ipsi, middle bars) or bilateral (bilat, right-hand bars) transection of the lingual nerve (open bars) or mandibular branch of the trigeminal nerve (filled bars). Error bars in this and subsequent figures: S.E.M. *Significantly different from pre (ANOVA, P,0.05). (C) Individual example. Shown are peristimulus-time histograms (PSTHs; bin width, 1 s) of a gustatory NTS unit’s responses to lingual application of 0.3 M sucrose (upward and downward arrows in upper bars indicate onset and cessation of stimulus) before (left-hand PSTH) and after ipsilateral (middle PSTH) and bilateral (right-hand PSTH) transection of the lingual nerve. Upper right-hand inset (in box) shows a series of 15 successively recorded action potential waveforms superimposed.
lation followed by reduced firing. Interestingly, its tastantevoked response exhibited a similar biphasic pattern of initial facilitation followed by depression (Fig. 3, righthand PSTH). Of the five NTS units not directly driven by central mandibular stimulation, tastant-evoked responses were increased in two and unaffected in three.
3.4. Depression of tastant-evoked NTS responses by peripheral mandibular nerve stimulation In eight NTS units (four citric acid, three sucrose, one NaCl), electrical stimulation of the peripheral trunk of the sectioned ipsilateral mandibular nerve consistently reduced
tastant-evoked responses to variable degrees, with an overall significant decrease (P50.022) that reached a nadir at 2–4 min post-stimulation and returned to pre-stimulation levels thereafter (Fig. 5).
3.5. Sympathetic stimulation Stimulation of the ipsilateral superior cervical sympathetic ganglion did not result in any significant (P50.798) change in tastant-evoked responses of six NTS units tested (two citric acid, three sucrose, one NaCl). Fig. 6A shows that the mean tastant-evoked responses of these units were unaffected following ganglionic stimulation (applied prior
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Fig. 3. Example of gustatory NTS unit driven by electrical stimulation of the central cut end of the mandibular nerve. PSTHs show, from left to right, the unit’s response to citric acid (upper bar with arrows), trigeminal stimulation alone (TG stim., at middle bar), and trigeminal stimulation (at right-hand bar) followed immediately afterward by citric acid. Note that TG stimulation excited the unit followed by inhibition (middle PSTH), and similarly facilitated and then inhibited the response to citric acid (right-hand PSTH). Upper inset shows superimposed action potential waveforms.
to trial 2). Since ganglionic stimulation had no effect, these data further illustrate the relative stability of tastant-evoked responses across repeated trials. Fig. 6B shows an individual example of the stable responses of a unit to repeated applications of citric acid.
4. Discussion The present study provides evidence for direct and / or indirect modulatory effects of the trigeminal nerve on gustatory processing by NTS neurons. Unilateral transection of the lingual or mandibular nerve resulted in a 20–30% decrease in the magnitude of tastant-evoked responses of NTS neurons. Furthermore, electrical stimulation of the central end of the sectioned mandibular nerve directly excited nine of 14 gustatory NTS neurons, consistent with the possibility that some trigeminal primary afferents may have direct excitatory projections to NTS. Central mandibular stimulation variably affected tastantevoked responses of NTS cells, with 43% showing an increase and 22% a decrease. Finally, electrical stimulation of the peripheral end of the sectioned mandibular nerve resulted in an |20% decrease in the tastant-evoked responses of gustatory NTS neurons. These findings are discussed in terms of possible underlying neural mechanisms and behavioral correlates.
Fig. 4. Effects of central trigeminal stimulation on tastant-evoked responses of NTS neurons. (A) Scatter plot as in Fig. 2A graphing each neuron’s response to the tastant before and after stimulation of the central cut end of the mandibular nerve. Filled symbols, response not affected; open symbols, response increased; shaded symbols, response reduced. (B) Bar graph plots mean responses vs. time for six units whose responses increased (by .1.96 S.D. of mean baseline level) after trigeminal nerve stimulation. Error bars, S.E.M. **Significantly different (ANOVA, P, 0.05) from control response (pre). (C) Individual example of NTS unit’s successive responses to NaCl relative to an episode of central trigeminal nerve stimulation (indicated by bar). Note the transient increase in NaCl-evoked response size following TG stimulation (2nd–4th PSTHs from left). Format as in Fig. 2C.
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Fig. 5. Depression of NTS tastant-evoked responses by electrical stimulation of the peripheral cut end of the mandibular nerve. Graph as in Fig. 4B, plotting mean tastant-evoked responses over time. *Significantly different from pre (P,0.05).
4.1. Reduced gustatory NTS neuronal responses after trigeminal nerve transection The presently observed reduction in the magnitude of NTS neuronal tastant-evoked responses following transection of the lingual or mandibular nerves might be explained by (a) non-specific consequences of nerve transection (e.g. injury discharge) which might produce acute peripheral changes that altered the chorda tympani’s sensitivity to tastants, as discussed later, (b) the elimination of trigeminal input evoked by tastant stimuli, or (c) removal of tonic trigeminal excitation of NTS (disfacilitation). Regarding the second possibility, citric acid at high (250–300 mM) concentrations excites lingual nerve [6] and trigeminal subnucleus caudalis (Vc) neurons [48] and induces oral irritation [12]; the lower concentration used presently (30 mM), while not irritating, may conceivably have activated lingual fibers [43]. Therefore, eliminating input from citric acid-sensitive trigeminal fibers that project into NTS might partly account for the reduced responsiveness of these neurons to this stimulus post-nerve transection. However, this explanation is less likely for NaCl which excites the lingual nerve [47] and central Vc neurons [7] at higher concentrations than used presently, as well as for sucrose which is generally considered not to excite trigeminal afferents. Another possibility is that trigeminal nerve transection removed a tonic excitation of NTS neurons, rendering them less excitable and hence reducing their tastant-evoked responses. Electrical stimulation of the central trunk of the sectioned mandibular nerve predominantly excited gustatory NTS neurons. If mandibular afferent fibers having
Fig. 6. Absence of effect of stimulation of superior cervical sympathetic ganglion on NTS unit tastant-evoked responses. (A) Bar graph plotting mean tastant-evoked responses of six units across trials of repeated tastant stimulation. The superior cervical sympathetic ganglion was electrically stimulated prior to trial 2. (B) Individual example of an NTS unit’s stable responses to repeated application of citric acid. The superior cervical sympathetic ganglion was electrically stimulated prior to the second stimulus. Inset shows superimposed action potential waveforms.
excitatory synapses in NTS were tonically active, this could result in tonic depolarization of NTS neurons. Transection of the lingual or mandibular nerves would then remove this tonic facilitation to result in a reduction (disfacilitation) in tastant-evoked responsiveness of NTS neurons as was presently observed. This mechanism is consistent with the clinical observation that patients with trigeminal disorders possibly associated with partial deafferentation exhibit higher taste detection thresholds as assessed by electrogustometry [4,16]. That transection of the lingual nerve innervating the tongue had a somewhat smaller depressant effect on tastant-evoked responses compared to mandibular transection (Fig. 2A) suggests that any tonic trigeminal facilitatory effect originates not just from lingual afferents but also from mandibular afferents
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innervating other tissues as well. However, transection of neither the mandibular nor the lingual nerves had any significant effect on the spontaneous firing rate of gustatory NTS neurons, arguing against the notion that these nerves provide a tonic facilitatory input to NTS.
4.2. Modulation of gustatory NTS neurons by central mandibular stimulation Electrical stimulation of the central cut end of the mandibular nerve directly excited a majority (nine of 14) of gustatory NTS units and variably affected their tastantevoked responses, with six facilitated, three reduced and five unaffected. These effects might be mediated via direct primary afferent input to NTS (see Introduction), as well as indirect projections to NTS from second- or higher-order neurons in brain stem trigeminal subnuclei [34]. Some trigeminal primary afferents release substance P or other neuropeptides from their central terminals [9,13]. Neurokinin 1 receptors, to which substance P preferentially binds, have been identified in the rostrolateral NTS [11] that receives trigeminal projections [19,26–28,31,49]. Microinjection of substance P into the NTS variably modulated responses of gustatory NTS neurons, with 48% showing an increase and 9% a decrease in firing [10] similar to the percentages of NTS neurons exhibiting increases and decreases in tastant-evoked responses following central mandibular stimulation observed here. We cannot completely rule out a possible parasympathetic effect on lingual blood flow which might indirectly affect NTS unit tastant-evoked responses when the central cut end of the mandibular nerve was stimulated. In the cat, electrical stimulation of the central cut end of the lingual nerve elicits increases in blood flow to oral structures [25] possibly via a parasympathetically-mediated vasodilation [24]. However, this should affect neuronal responses consistently, while we observed mixed effects following central mandibular stimulation. In this regard, a parasympathetically-mediated increase in salivary flow might indirectly affect tastant-evoked responses [17], but any increase in salivary volume would be small compared to the volume of tastant applied by constant flow and hence unlikely to appreciably change the tastant concentration.
4.3. Depression of gustatory NTS neurons by peripheral mandibular stimulation In contrast to the mixed but predominantly excitatory effect of central mandibular nerve stimulation, electrical stimulation of the distal cut end of the mandibular nerve had a predominantly depressant effect on gustatory responses of NTS neurons. It was shown previously that electrical stimulation of the lingual nerve [51], as well as intraoral capsaicin [41], suppressed NaCl-evoked responses in chorda tympani fibers. We have recently reported that lingual capsaicin also reduces the tastant-
evoked responses of NTS neurons largely through a peripheral mechanism, since capsaicin depression of tastant-evoked NTS responses was equally robust following complete bilateral trigeminal ganglionectomy [46]. One possibility is that trigeminal nerve stimulation evokes the release of substance P and / or other neuropeptides from the peripheral nerve endings in the tongue. Peripheral nerve fibers of trigeminal origin are found in high density in and around taste buds [14,35,37,54]. It was proposed that the local release of neuropeptides from trigeminal nerve endings might decrease the sensitivity of taste receptor cells, explaining capsaicin and lingual nerve stimulationevoked reductions in responses of chorda tympani fibers to NaCl [41,46,51]. However, given that sympathetic nerve fibers are present in the trigeminal ganglion [33], it is also possible that activation of sympathetic reflexes (e.g. blood flow changes) might contribute to the suppression of tastant-evoked NTS responses following electrical nerve stimulation. Prior studies have shown that altering blood pressure can modulate the magnitude of tastant-evoked responses in chorda tympani fibers [21,22]. An indirect sympathetically-mediated effect is mitigated by our present finding that electrical stimulation of the superior cervical sympathetic ganglion did not significantly alter tastantevoked responses of NTS neurons. It is interesting to note that in our previous study, lingual capsaicin application suppressed NTS taste responses to about 60% of pre-capsaicin levels [46], an effect that was greater than that of peripheral mandibular nerve stimulation (to about 80% of control). This suggests that capsaicin is more effective than electrical nerve stimulation to evoke peripheral neuropeptide release and / or to engage other mechanisms to suppress gustatory transmission [46]. It is conceivable that local alterations in lingual temperature may have contributed to the observed effects. Tastantevoked responses are thermally sensitive [36] and a prior study, using the same stimulation parameters used here, noted an |5 8C increase in rat tongue temperature [51]. However, such a thermal effect would be reduced to some extent in our study, since tastants were delivered by constant flow at room temperature such that the fluid volume on the tongue would ‘clamp’ the tongue temperature at a fairly constant level.
4.4. Behavioral correlates Interactions between cranial nerves innervating the tongue have been documented before [5], and several groups have proposed that the chorda tympani normally inhibits activity in the glossopharyngeal nerve [18,29]. This interaction is presumed to preserve taste function when damage to the chorda tympani and / or anterior portion of the tongue occurs [18,29]. Taste and somatosensory stimuli are invariably linked during mastication and swallowing. Because taste conveys invaluable information regarding the acceptability of foods, it seems
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reasonable that somatosensory information carried by trigeminal afferents into the NTS would modulate gustatory activity to make it more physiologically meaningful. For instance, it has been reported that the trigeminal nerve is sensitive to quinine [30,43], and one may speculate that enhanced trigeminal activity could potentiate NTS gustatory responses to quinine. Further studies are needed to elucidate the behavioral significance of the modulatory effects of the trigeminal nerve on gustatory processing described here.
[12]
[13]
[14]
[15]
Acknowledgements This work was supported by grants from the International Association for the Study of Pain, the Institut Francais pour la Recherche Odontologique, the California Tobaccorelated Disease Research Program ([ 10DT-0197 and 11RT-0053), and the National Institute of Dental and Craniofacial Research ([ DR13685). The authors gratefully acknowledge the excellent histological assistance of M. Iodi Carstens.
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