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INTRODUCTION |
Despite the widespread consumption of food spices and tobacco products that contain chemicals that irritate oral and ocular mucosa, little is known about the central neural mechanisms underlying the resulting irritant sensations. Parker (1912)
proposed the concept of a "common chemical sense" elicited by noxious chemicals. This was mediated by free nerve endings and served a protective function. A variety of chemicals elicit irritation or pain when delivered to the oral or ocular mucosa (reviewed in Green and Lawless 1991; Green et al. 1990) (see DISCUSSION). Sensations from some of these irritants (capsaicin, NaCl, citric acid, cinnamic aldehyde, and menthol) are cross-desensitized by capsaicin (Cliff and Green 1996; Dessirier et al. 1997; Green 1991, 1996; Gilmore and Green 1993), suggesting that the irritation is mediated partly by a common population of capsaicin-sensitive fibers. It is currently uncertain whether different qualities of oral or ocular irritation can be discriminated (see DISCUSSION) or to what extent central trigeminal neurons can be activated by one or more irritant chemical. The present study was undertaken to address this latter question.
Irritant chemicals in the oral cavity presumably activate chemosensitive nociceptors the free endings of which are located in the mucosal epithelium and lamina propria (Holland 1984). Irritants contacting the ocular surface activate epithelial free nerve endings of nociceptors having thinly myelinated or unmyelinated fibers (MacIver and Tanelian 1993a,b). Single-fiber recordings from lingual (Bryant and Moore 1995; Hellekant 1965; Komai and Bryant 1993; Lundy and Contreras 1994; Okuni 1978; Sostman and Simon 1991; Wang et al. 1993) or ciliary nerves (Belmonte and Giraldez 1981; Belmonte et al. 1991; Chen et al. 1995, 1997; Gallar et al. 1993; MacIver and Tanelian 1993b; Tanelian 1991), indicate that polymodal nociceptor and mechanically insensitive afferents can respond to irritant chemicals (acetylcholine, nicotine, NaCl and other salts, acid, capsaicin, and CO2) although the degree of chemoselectivity of individual fibers is currently uncertain. Recent patch-clamp studies of small-diameter trigeminal ganglion neurons (Liu and Simon 1994, 1996a-c; Liu et al. 1993, 1997) suggest that at least some are capable of responding to more than one class of irritant chemical.
Afferent fibers innervating the tongue pass via the lingual nerve of the mandibular division, whereas afferent fibers from the cornea-conjunctiva pass via the ciliary nerve in the ophthalmic division, to terminate in brain stem trigeminal subnuclei caudalis, interpolaris, oralis, and principalis in a somatotopically organized manner with the head inverted and facing medially (Arvidsson and Gobel 1981; Darian-Smith et al. 1963; Dostrovsky and Hellon 1978; Hamilton and Norgren 1984; Hayashi 1985; Jacquin et al. 1983, 1986, 1988; Kruger and Michel 1962; Lu et al. 1993; Marfurt 1981; Marfurt and Del Toro 1987; Meng and Bereiter 1996; Nord 1968; Panneton and Burton 1981; Renehan et al. 1986; Rowe and Sessle 1972; Shigenaga et al. 1986a,b; Schults 1992a; Takemura et al. 1991; Torvik 1956; van Ham and Yeo 1996 reviewed in Norgren 1984). Fibers traveling in the chorda tympani to the nucleus of the solitary tract also may contribute to irritant sensations (Norgren 1984). Electrophysiological studies indicate that a substantial fraction of neurons in subnucleus caudalis responds differentially or exclusively to noxious stimulation of intraoral tissue, cornea or face (e.g., Amano et al. 1986; Bushnell et al. 1984; Chiang et al. 1994; Hu 1990; Hu et al. 1981; McHaffie et al. 1994; Meng et al. 1997; Mosso and Kruger 1973; Nagano et al. 1975; Nishida and Yokota 1991; Pozo and Cervero 1993; Price et al. 1976; Raboisson et al. 1995; Renehan et al. 1986; Sessle et al. 1981, 1986; Yokota 1975; Yokota and Nishikawa 1977, 1980; Yu et al. 1993; reviewed in Schults 1992b). Neurons in the interstitial (paratrigeminal) nuclei also respond to noxious stimuli, and it was suggested that these may represent a rostral extension of lamina I of subnucleus caudalis (Hayashi and Tabata 1989b; Schults 1992b). Furthermore, neurons in more rostral trigeminal subnuclei interpolaris (Hayashi et al. 1984; Ohya 1992) and oralis (Dallel et al. 1990, 1996; Hayashi and Tabata 1989a; Hu and Sessle 1984; Jacquin and Rhoades 1990; Raboisson et al. 1991; Sessle and Greenwood 1976) also respond to noxious orofacial stimuli although the incidence of such nociresponsive neurons is lower compared with caudalis. A primary role for subnucleus caudalis in signaling pain is supported by recent immunohistochemical data showing that neurons expressing c-fos after noxious orofacial stimulation are located in somatotopically appropriate regions of subnucleus caudalis but not more rostral trigeminal subnuclei (Anton et al. 1992b; Bereiter et al. 1994; Carstens et al. 1995; Coimbra and Coimbra 1994; Lu et al. 1993; Mineta et al. 1995; Meng and Bereiter 1996; Strassman and Vos 1993).
To date there have been relatively few studies of the responses of trigeminal neurons to irritant chemicals in general (Amano et al. 1986; Ebersberger et al. 1997; Hu et al. 1992; Meng et al. 1997; Mosso and Kruger 1973; Peppel and Anton 1993; Raboisson et al. 1991, 1995; Yu et al. 1993) and, to our knowledge, none concerning neuronal responses to application of irritant chemicals onto the surface of the tongue. We recently reported that application of different irritant chemicals (nicotine, capsaicin, piperine, and histamine) onto the dorsal tongue resulted in a similar distribution of c-fos-immunoreactivity in the superficial layers of the dorsomedial aspect of trigeminal subnucleus caudalis, in addition to other brain stem areas (Carstens et al. 1995). Others have reported that noxious mechanical or chemical stimulation of the cornea produces two distributions of c-fos-immunoreactivity, one in the ventrolateral aspect at the transition of rostral caudalis to interpolaris, and a more caudal distribution in the ventrolateral dorsal horn of the upper cervical spinal cord (Bereiter and Bereiter 1996; Bereiter et al. 1994; Lu et al. 1993; Martinez and Belmonte 1996; Meng and Bereiter 1996; Strassman and Vos 1993). A limitation of the c-fos method is that it cannot distinguish whether one and the same neuron is activated by different chemicals or if there are separate populations of "chemospecific" neurons grouped near one another with each responding only to one chemical. For this reason, electrophysiological experiments were undertaken to determine if single neurons in superficial dorsomedial caudalis can respond to application onto the tongue of a variety of irritant chemicals that are presumed to act via different peripheral transduction mechanisms (Brand and Bryant 1994; M. Kress and P. W. Reeh, unpublished data). Our aim was to sample a variety of irritant chemicals, some of which act at specific molecular receptors (e.g., histamine, capsaicin, serotonin, and nicotine), whereas others have nonspecific or unknown effects on the nociceptor terminal membrane. For comparison, we similarly tested if neurons in the ventrolateral aspect of caudalis receiving input from the cornea-conjunctiva respond to different irritant chemicals applied to the ocular surface. Abstracts of this work have appeared elsewhere (Carstens et al. 1996; Kuenzler et al. 1996).
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METHODS |
Surgery
Experiments were conducted using 30 adult male Wistar rats (300-450 g). Anesthesia was induced with thiopental (120 mg/kg ip). Supplemental doses of thiopental (20-60 mg/kg ip) were administered as necessary to maintain a constant level of anesthesia as assessed by areflexia, absence of any organized movements (e.g., of the tongue), and absence of heart rate changes (as monitored by electrocardiogram) on noxious stimulation. A tracheotomy was performed, and a catheter was placed in the jugular vein for infusion of isotonic saline and paralytic agent. Scopolamine (50 mg/kg sc) was given. Core temperature was monitored and maintained at ~37°C by a feedback-controlled infrared lamp. During recording, the animals were paralyzed (Pancuronium, bolus injection of ~0.5 mg/0.25 ml iv) and mechanically ventilated at a rate and tidal volume sufficient to maintain end-tidal CO2 (monitored with a Datex infrared CO2 analyzer) at 4-5%. The anesthetic level was checked periodically when the effect of the paralytic agent waned.
The upper cervical spine and occipital bone were exposed by midline incision, and the base of the cerebellum, lower brain stem, and C1 spinal cord exposed by removal of the atlas and caudal-most part of the occipital bone. The animal was placed in a stereotaxic frame, and the head fixed in a ventroflexed position. The upper cervical spine was rigidly held in place with a vertebral clamp. The exposed surface of the brain stem was covered with agar. A small opening was made in the hardened agar to expose the brain stem, which then was bathed in warmed isotonic saline.
Single-unit recording
The recording microelectrode was advanced into the brain stem in 5-µm steps using a piezoelectric microdrive. In most experiments, a Teflon-insulated tungsten microelectrode (WPI; ~10 M
) was used, while in a few experiments a glass-coated carbon-fiber microelectrode was used. Extracellular single-unit activity was amplified and displayed by conventional means and fed via a Microstar analog-digital converter to a computer. Unitary action potentials were discriminated, and instantaneous frequency sampled continually, using software developed in Erlangen (Forster and Handwerker 1990).
Recordings were made from separate unit populations: units in superficial layers of the dorsomedial trigeminal subnucleus caudalis that responded to pressure and pinch stimuli applied to the tip or side of the ipsilateral tongue ("tongue" units) and units in ventrolateral caudalis that responded to tactile stimulation of the cornea-conjunctiva ("corneal-conjunctival" units). In 11 rats, only units with tongue input were studied, whereas in 17 rats, a unit with input from tongue as well as another unit with input from the cornea-conjunctiva were studied. In two rats, only units with corneal input were studied. We believe it is unlikely that the prior recording from a tongue unit influenced the responses of the subsequently recorded corneal units (or vice versa) because units with convergent afferent input from the tongue and cornea were never observed and because we detected no obvious differences in data obtained from the experiments in which cornea units were recorded first compared with those in which tongue units were recorded first. In about one-half (10/19) of experiments with corneal unit recordings, separate units with input from the left and right cornea were recorded. We also did not detect any marked differences in data obtained from the first compared with second corneal unit recorded in the same experiment. Before recording corneal-conjunctival units on one side, the ocular surface was covered with an ointment (Bepanthen, Roche) to prevent desiccation.
The search for units with input from the tongue was restricted to the area ~0-2 mm caudal to the obex and ~1.5 mm lateral to the midline, based on earlier c-fos immunohistochemical mapping studies (Carstens et al. 1995; Strassman and Vos 1993). Units responsive to mechanical stimulation of the tongue were identified readily at depths ranging from 50 to 300 µm, and no deeper than 600 µm, below the surface of the brain stem. To search for units responsive to mechanical stimulation of cornea-conjunctiva, microelectrode penetrations were made 1-3 mm caudal from obex and 2.5 mm lateral to the midline and at depths ranging from 1-2.5 mm below the medullary surface.
Characterization of tongue units
The mouth was held open to access the dorsal anterior tongue, which was frequently moistened with isotonic saline to prevent desiccation. Only units that responded to mechanical (pressure, pinch) stimulation of the tongue, and additionally to noxious thermal stimulation (topical application of hot water at 48-54°C water to the dorsal tongue), were selected for further study. For units with no spontaneous activity, any stimulus-evoked discharge was considered to be a response. For units with spontaneous activity, a response was generally considered as a two- to threefold increase in firing rate during stimulus presentation; this subjective definition was borne out by subsequent statistical analysis (see further text). Units' mechanosensitive receptive fields were mapped approximately using pressure-pinch stimulation with forceps or small arterial clamps exerting different forces and with von Frey hairs. The extent of lingual mechanical receptive fields was difficult to map precisely because the tongue was not stabilized. All units were additionally tested for sensitivity to cooling the tongue with a cotton ball cooled to ~4°C with an inert cold spray used routinely in dentistry (Kältespray, Schein-Dentina); an uncooled cotton ball served as a mechanical control stimulus. Units were classified as nociceptive-specific if they responded only to noxious levels of pressure-pinch stimuli (as judged by application of the same stimulus to the experimenter's tongue) and did not respond to the cold stimulus. There were classified as wide dynamic range (WDR) type if they responded to the cold stimulus and/or to nonnoxious mechanical pressure. Usually only one unit with tongue input was studied per animal; in four cases a second unit on the opposite side also was studied.
Characterization of corneal-conjunctival units
Mechanical brush, tap, and blunt pressure stimuli delivered to the cornea-conjunctiva and periorbital tissue were used to search for units. When a responsive unit was isolated, its mechanical receptive field was mapped more completely with graded von Frey hairs, and responsiveness to cooling was tested by applying a cold cotton ball to the eye. Only units the mechanical receptive field of which include the cornea and that additionally responded to noxious thermal stimulation of the cornea-conjunctiva by topical application of hot water (48-54°C) onto the surface of the eye (i.e., WDR type), were selected for further study.
Chemical stimulation of the tongue
Chemicals were applied topically by syringe onto the dorsal surface of the anterior tongue in a standard volume of 0.1 ml. The fluid volume covered an area of ~1 mm in diameter on the tip of the tongue bilaterally. In early experiments, a strip of Parafilm was placed underneath the tongue to prevent chemicals from reaching underlying tissue. However, because the head was ventroflexed, excess fluid dripping off of the dorsal surface of the tongue did not visibly contact tissue beneath the tongue so that the Parafilm strip was not used in later experiments. Each chemical was left on for 60 s, after which the tongue was rinsed with isotonic saline (0.9%). All chemicals were delivered at room temperature to avoid any confounding effect of cooling in the units that were cold sensitive. In a few cases, we also delivered chemicals continually at a constant flow rate and did not observe any marked prolongation in response duration, although this requires further investigation.
Chemical stimulation of cornea-conjunctiva
A volume of 0.1 ml of each noxious chemical was instilled into the eye by syringe. After 60 s, the eye was rinsed in a similar manner with isotonic saline (0.9%). No attempt was made to stimulate the cornea in isolation, so it is assumed that instilled chemicals activated receptors in ocular mucosa of the cornea and/or conjunctiva including the eyelid inner surface. The instilled fluid volume was held in place by surface tension, and no fluid was observed visibly to contact surrounding skin. Isotonic saline per se did not excite units. However, many units that were sensitive to mechanical stimulation of the cornea-conjunctiva or eyelid responded during physical application of the saline or noxious chemical due to direct activation of mechanoreceptors. These mechanically evoked discharges were always brief and restricted to the stimulus period (<2 s) and were in all cases readily distinguishable from chemically evoked responses that occurred later. When present, the mechanically evoked component of the response was subtracted from longer-latency discharges in analyzing unit responses to noxious chemicals.
Chemicals
The following chemicals were used routinely: capsaicin (0.001-1% = 3.3 × 10
2 to 3.3 × 105 M; diluted in dH2O from a stock solution of 1% in 80% ethanol; Sigma, Fluka), ethanol (EtOH; 15-80%, Merck), histamine (0.01-10% = 9 × 101 to 9 × 104 M in 0.9% NaCl; Sigma), mustard oil (allyl-isothiocyanate, 4-100% = 4 × 101 to 10 M, direct or diluted in paraffin oil; Merck), NaCl (0.5-5 M in dH2O), nicotine (0.01-10% = 6 × 101 to 6 × 104 M in 0.9% NaCl; Sigma), buffer solutions at preset pH values (pH range 1-6; Fisher Scientific), piperine (0.01-1% = 3.5 × 102 to 3.5 × 104 M, diluted in dH2O from a stock solution of 1% in 80% ethanol; Sigma), and serotonin 5-hydroxytryptamine (5-HT; 0.3-3% = 1.4 × 101 to 1.4 × 102 M in 0.9% NaCl; Sigma). In four experiments commercially available carbonated water (Cascada, pH 6.1) was used. Finally, in many experiments the H1 receptor antagonist cetirizine (Zyrtec, 0.1-1%; direct or diluted in 0.9% NaCl; UCB Chemie) (Simons and Simons 1991) or the nicotinic antagonist mecamylamine (0.1% = 4.9 × 105 M, in 0.9% NaCl; Sigma) also were used. The pH of all solutions except acidified buffer and carbonated water was neutral.
Sequential chemical stimulation
To determine if individual units respond to different chemicals delivered sequentially, it was imperative to determine if a given chemical induced long-lasting changes in the excitability of chemosensitive receptors in the tongue or ocular surface. Pilot experiments revealed that sufficiently high concentrations of capsaicin, piperine and mustard oil often desensitized the tongue, such that the unit no longer responded to subsequent application of any chemical. None of the other chemicals tested produced a marked desensitization. Therefore the different chemicals were delivered in a pseudorandom sequence except that capsaicin, piperine, and/or mustard oil were tested last. However, because later experiments focused on the effect of the H1 antagonist applied to the tongue, histamine was the first chemical tested in the majority (69%) of units with afferent input from the tongue.
Experimental design and data analysis
For each chemical tested, we delivered different concentrations to establish a dose-response relationship and/or a constant concentration repeatedly, to check for tachyphylaxis or sensitization. Chemicals were delivered at a 5-min interstimulus interval, which was chosen as a compromise to test as many of the 10 chemicals as possible for each unit. Generally, each chemical was delivered in ascending order of concentration so that the dose-response relationship was determined first. If the unit responded robustly at a given concentration, the chemical was then delivered successively at that concentration two to four times to check for tachyphylaxis. After collecting data for one chemical, the next chemical was similarly tested. In some cases, tachyphylaxis was tested first using a suprathreshold concentration and the dose-response relationship was either determined later or not at all. Thus we could not obtain both dose-response and tachyphylaxis data for all units. For a given unit, we attempted to test as many of the 10 chemicals as possible in this manner. Data for each chemical were pooled to generate profiles of the time course of mean responses (Figs. 4 and 5), population dose-response relationships, and response levels over repeated trials (Figs. 6 and 9). Responses to a given chemical at suprathreshold concentration were averaged, and a paired t-test compared the average firing rate before the chemical with the average firing rate at 1-s intervals after chemical application. There was a degree of variability in absolute response magnitude across neurons. Therefore the population dose-response data, and mean responses across application trials, for each chemical were subjected to a nonparametric van der Waerden analysis of overall treatment effects, followed by post hoc comparisons among treatment levels; P < 0.05 was accepted as significant.
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| FIG. 4.
Time course of responses of dorsomedial caudalis units to application of different chemicals to the tongue. Shown are averaged PSTHs (binwidth: 1 s) of group responses to application of each of the indicated chemicals at a suprathreshold concentration (1 or 10% for histamine, 1 or 10% for nicotine, 2.5 or 5 M for NaCl, 25 or 50% for EtOH, 3% for 5-HT, pH 1-3 for acid, 0.001-0.1% for capsaicin, and 7.5-25% for mustard oil). Numbers above PSTHs indicate number of units. Error bars: SD; * Significantly different from mean response before chemical stimulus application (P < 0.05, paired t-test).
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| FIG. 5.
Time course of responses of ventrolateral caudalis units to application of different chemicals to the cornea-conjunctiva. Averaged PSTHs as in Fig. 4 for group responses to application of each of the indicated chemicals to cornea-conjunctiva. Numbers above PSTHs indicate number of units. Error bars: SD; * Significantly different from mean response before chemical stimulus application (P < 0.05, paired t-test).
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| FIG. 6.
Dose-response relationships and tachyphylaxis: tongue units. Left: dose-response relationship. Each graph shows individual (thin lines) and mean (thick line with SE) dose-response curves for the indicated irritant chemical. Responses (total imp/60 s period of chemical application) are plotted vs. concentration; SA on the x axis indicates spontaneous activity. * Mean response significantly greater (P < 0.05, van der Waerden). Right: tachyphylaxis. Each graph plots individual (thin lines) and mean (thick line with SE) unit responses to application of the indicated chemical at 1 suprathreshold concentration. * Significantly smaller than trial 1 (P < 0.05, van der Waerden). SA, spontaneous activity. A and B: histamine; C and D: nicotine; E and F: NaCl; G and H: EtOH; I and J: serotonin (5-HT); K and L: acidified buffer at fixed pH; M and N: capsaicin; O and P: piperine; Q and R: mustard oil.
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| FIG. 9.
Dose-response relationships and tachyphylaxis: cornea-conjunctiva units. Left: dose-response relationship (format as in Fig. 6). * Mean response significantly greater (P < 0.05, van der Waerden). Right: tachyphylaxis (format as in Fig. 6). * Significantly smaller than trial 1 (P < 0.05, van der Waerden). A and B: histamine; C and D: nicotine; E and F: NaCl, G and H: EtOH; I and J: 5-HT; K-M: acidified phosphate buffer at fixed pH; N and O: capsaicin; P and Q: mustard oil; R and S: piperine.
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We investigated the effect of the H1 antagonist (tongue units only) and the nicotinic antagonist (tongue and corneal-conjunctival units) as follows. To test the effect of the H1 antagonist, histamine (10%) was delivered to the tongue at least three times at 5-min interstimulus intervals. Thirty seconds before the next scheduled histamine stimulus, cetirizine was delivered to the tongue in an identical manner. Histamine continued to be delivered at the 5-min interstimulus interval to evaluate the time course of any effect of cetirizine on histamine-evoked responses. A similar paradigm was followed to determine the effect of mecamylamine on nicotine-evoked responses of tongue or corneal-conjunctival units. In some cases, the H1 antagonist was tested similarly against nicotine-evoked responses and mecamylamine against histamine-evoked responses. Data were pooled and mean responses before and after application of the antagonist were compared using a paired t-test.
For experiments in which capsaicin was tested on tongue units, we usually observed an increase in spontaneous activity after application of capsaicin at a suprathreshold concentration. In these cases, we recorded the spontaneous firing for 
1 h after the capsaicin stimulus. Data were pooled and mean spontaneous firing levels at different times postcapsaicin were compared with the precapsaicin baseline level using a paired t-test.
Histology
At the conclusion of successful recordings, an electrolytic lesion was made at the recording site by passing current (6 V DC) through the microelectrode. In the few experiments using carbon-fiber microelectrodes, the exact location of the electrode penetration was noted, a tungsten microelectrode was inserted at that site to the same depth, and an electrolytic lesion was made. At the conclusion of the experiment, the animal was killed by overdose of thiopental, and the brain stem removed and postfixed in 10% formalin. The brain stems were cut in 50-µm frozen sections, collected on glass slides, counterstained, and examined under the light microscope. Sections containing the lesion were drawn by camera lucida. Lesion sites were collectively plotted onto representative brain stem sections (Fig. 1).
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| FIG. 1.
Unit recording sites.  , locations of histologically recovered recording sites (i.e., lesions) in superficial dorsomedial trigeminal subnucleus caudalis for units responsive to stimulation of the ipsilateral tongue.  , locations of recording sites for units responding to stimulation of ipsilateral cornea-conjunctiva. Sites are plotted on representative sections through the caudal medulla. ···, approximate lamina II-III border. Cu, cuneate n.; Gr, n. gracilis; ION, inferior olivary n.; LRN, lateral reticular n.; NTS, n. solitary tract; Pyr, pyramid; Px, pyramidal decussation; Vc, trigeminal n. caudalis.
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RESULTS |
Unit sample
TONGUE UNITS.
Each of the 32 units responded to mechanical (pressure-pinch) stimuli and noxious heating of the tongue, 60% responded to cooling, and all but one responded additionally with a clear increase in firing rate to at least one chemical (Table 1). The majority of units exhibited low (<1-2 Hz) or no background firing, whereas the remainder were spontaneously active at rates 8 Hz (maximum 500 imp/60 s). The mean spontaneous firing rates for all tested units are plotted in Fig. 6. Mechanically sensitive receptive fields almost always included the tip of the tongue as well as more posterior and lateral areas of the tongue ipsilaterally. Responses usually appeared to be evoked by pinching the tip of the tongue bilaterally, although it was difficult to ascertain the precise extent of the mechanical receptive fields because the tongue was not stabilized. The majority of units (55%) had mechanical receptive fields solely on the tongue, whereas the remainder responded additionally to pressure or pinch stimuli delivered to the lower lip and/or point of the chin ipsilaterally. One unit additionally responded to pinching the corner of the mouth. Most units appeared to respond to bilateral stimulation of the tip of the tongue when it was within the receptive field (Figs. 2 and 10A). In general, units with receptive fields including tongue and chin tended to be located caudal to those with input only from the tongue. Eighty-one percent (26/32) of the units were categorized as WDR because they responded to nonnoxious mechanical stimuli and/or innocuous cooling of the tongue as well as noxious heat, whereas the remainder (19%) were categorized as nociceptive-specific because they responded only to noxious pinch and heat stimuli. The incidence of responsiveness of nociceptive-specific units to different chemicals was comparable with that of WDR units; hence, data from both unit classes are presented together.
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TABLE 1.
Incidence of individual dorsomedial caudalis (tongue) unit responses to application of various chemicals to the tongue
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| FIG. 2.
Example of responses of individual caudalis unit to application of different stimuli to the tongue. Each row of peristimulus-time histograms (PSTHs; binwidth: 1 s) shows responses to application of 1 chemical. All PSTHs have the same x axis (time; shown in bottom PSTHs) and y axis (response; shown in left PSTHs). Stimuli (0.1 ml) were delivered topically to the dorsal tongue (at arrow) and left on for 60 s, followed by rinse with 0.9% saline. First 7 rows, from left to right: responses to increasing doses (1st 3 PSTHs) and to repeated application at 1 dose (last 3 PSTHs). Row 8: responses to repeated mustard oil (MO); bottom row: responses to physical stimuli (left), piperine (middle), and carbonated water (right). Hist, histamine; pH, acidified phosphate buffer at indicated pH value; EtOH, ethanol (50%); cap, capsaicin; MO, mustard oil. Bottom left inset: receptive field (black) on ipsilateral tongue and chin. Bottom right inset: recording site (filled circle) on drawing of brain stem section.
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| FIG. 10.
Selective antagonism of caudalis unit responses to histamine and nicotine applied to the tongue. A, top: individual unit's response to histamine (10%) before (left), 30 s after topical application of the H1 antagonist cetirizine (Zyrtec, 1%; middle), and 5 min later (right). Note marked reduction in histamine-evoked response by cetirizine. A, middle: same unit's response to nicotine (1%; left) and lack of effect of cetirizine on nicotine response (right). A, bottom: same unit's response to nicotine was reduced markedly 5 min after topical application of mecamylamine (0.1%; middle) with partial recovery after 1 h (right). Inset: receptive field on ipsilateral tongue tip and chin. B, top: initial response of a different unit to nicotine (1%; left), and marked reduction of its response to nicotine 30 s (middle) and 20 min (right) after application of mecamylamine (0.1%) to the tongue. B, bottom: responses of this unit to histamine (10%), before (left) and 30 s after (right) application of mecamylamine (0.1%) to tongue. Histamine-evoked response was not reduced by mecamylamine.
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CORNEAL-CONJUNCTIVAL UNITS.
A total of 31 units responsive to tactile and noxious thermal stimulation of the cornea-conjunctiva and periorbital tissue was recorded. Mechanical receptive fields of some units were restricted to the cornea only (n = 1) or cornea-conjunctiva and eyelids (n = 13), whereas the remainder had larger receptive fields extending anteriorly into facial skin. Seven of the latter units had receptive fields incorporating hairy skin on the snout; we did not test if any of these units responded to noxious chemical stimulation of the nasal mucosa as has been described previously for units with similar receptive fields (Peppel and Anton 1993).
Stimulation of the cornea with a moving brush or filament was particularly effective in exciting units. When perpendicular punctate stimuli were delivered at one corneal site, fairly high von Frey bending forces (16 to >256 g), or pin-prick stimuli, were needed to evoke a response. In contrast, von Frey thresholds for evoking responses from the eyelid were extremely low (<1 mN). The units with larger facial receptive fields responded to blowing or light brushing of fur as well as to noxious cutaneous pin-prick stimuli. A majority of units tested (19/28) responded to cooling of the cornea-conjunctiva. In addition to their responses to mechanical and cooling stimuli, all units responded to noxious heat and were thus classified as WDR.
Eighteen of the units did not exhibit any spontaneous firing, while the remainder fired spontaneously. Ten units fired spontaneously at a low rate (1 Hz), whereas 3 fired at rates of 3-10 Hz.
Unit recording sites
TONGUE UNITS.
Recording sites were histologically recovered in most (84%) experiments, and are shown in Fig. 1 (). Virtually all were located in the most superficial layer of the dorsomedial trigeminal nucleus caudalis, ipsilateral to the receptive field, in a distribution indistinguishable from that of c-fos-immunoreactive cell nuclei after noxious chemical stimulation of the tongue (Carstens et al. 1995).
CORNEAL-CONJUNCTIVAL UNITS.
Recording sites were histologically recovered for 25 units and are shown in Fig. 1 (). Four were located superficially, and the remainder were located in deeper layers of the ventrolateral trigeminal nucleus caudalis, ipsilateral to the unit's receptive field.
Responses to different noxious chemicals
TONGUE UNITS.
All but one unit responded to application of at least one noxious chemical to the tongue, and most units responded to several different chemicals (Table 1). Overall, the 32 units responded to 75.3% of the different chemicals tested, and nearly one-third responded to all chemicals (10) applied. Thus 87.5% of the units tested responded to histamine, 81% to nicotine, 74% to NaCl, 61% to 5-HT, 89% to acid, 80% to ethanol, 75% to capsaicin, 55.5% to piperine, and 50% to mustard oil. The percentages for piperine and mustard oil are probably an underrepresentation because these chemicals were almost always tested after prior application of capsaicin [Table 1, (
)], which frequently desensitized the tongue (see further text).
Unit responses to application of a given chemical were not markedly affected by prior application of a different chemical, except for capsaicin, piperine, and mustard oil, which often appeared to desensitize the tongue. We determined the number of chemicals evoking responses in neurons that were grouped according to the chemical that they were first exposed to. The 22 units tested first with histamine subsequently responded to 82.4% of up to eight additional chemicals tested. Similarly, four units tested first with 5-HT subsequently responded to 88.4%, three with NaCl to 73.7%, and two with acid to 85.7% of the additional chemicals tested. One unit tested initially with nicotine responded to both of two additional chemicals.
An example of one unit's responses to a variety of different chemicals is shown in Fig. 2. Each row shows peristimulus time histograms (PSTHs) of the unit's responses to a given chemical, arranged from left to right by increasing concentration (1st 3 PSTHs in row) and by repeated trials at one concentration (last 3 PSTHs in row). The bottom row shows, from left to right PSTH, the unit's responses to physical stimuli, piperine, and carbonated water. This unit had a receptive field on the ipsilateral tip of the tongue and chin (Fig. 2, bottom left inset) and responded strongly to noxious heating of the tongue but only weakly to pressure and did not appear to respond appreciably to cooling (Fig. 2, bottom left). Most importantly, this unit responded to each chemical tested, as indicated by the middle column of PSTHs. Responses evoked by each of the chemicals were of comparable magnitude, but response duration appeared to be briefer for histamine, nicotine, ethanol, and carbonated water compared with the other chemicals. Furthermore, response magnitude increased in a dose-related manner for each chemical tested (1st 3 PSTHs in rows 1-7). Successive responses to repeated suprathreshold applications of 5-HT, nicotine, capsaicin, and mustard oil decreased markedly across trials (3 righthand PSTHs in rows 1, 4, 7, and 8, respectively), whereas successive responses to the other chemicals decreased less or not at all. The unit's recording site was in dorsomedial caudalis (Fig. 2, bottom right inset).
CORNEAL-CONJUNCTIVAL UNITS.
Most of these units responded to a majority or all of the tested chemicals, whereas only two were unresponsive (Table 2). Overall, the 31 units responded to 65% of up to nine chemicals tested per unit (116/179 chemical stimulus applications). Table 2 provides an overview of unit responsiveness to the various chemicals. The percentages of units responding to each chemical are as follows: nicotine (88%), capsaicin (82%), EtOH (79%), acid (70%), NaCl (67%), piperine (61.5%), mustard oil (58%), histamine (46%), and 5-HT (25%).
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TABLE 2.
Incidence of individual ventrolateral caudalis (corneal-conjunctival) unit responses to application of various
chemicals to cornea-conjunctiva
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An individual example of a unit's responses to various chemicals applied to the cornea-conjunctiva is shown in Fig. 3. This unit had a cutaneous receptive field that encompassed the eye and part of the lateral face (bottom left inset, hatching) and responded to noxious heat and innocuous cold and pressure stimuli applied to the ocular surface (Fig. 3, bottom left PSTH). The PSTHs in the middle column and in the bottom row show the responses of this unit to application of each of the nine chemicals tested. Note that this unit usually gave a brief high-frequency discharge when the chemical was applied (at arrows), and again 60 s later when the saline rinse was applied; this represents a response to mechanical stimulation by the fluid drop. The chemically evoked response occurred after the initial mechanical response. This unit gave the largest responses to histamine, nicotine, NaCl (1st 3 rows), ethanol (5th row), and mustard oil (middle PSTH in bottom row). The three left-hand columns of PSTHs in the first three rows show that the unit's responses increased with concentration of histamine, nicotine, and NaCl. The three right-hand columns show that the unit's responses generally declined on repeated application of most of the chemicals, with the exception of NaCl (3rd row).
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| FIG. 3.
Example of responses of individual caudalis unit to application of different stimuli to cornea-conjunctiva. Format as in Fig. 2. Chemical stimuli (0.1 ml) were instilled topically into the eye and left on for 60 s, followed by rinse with 0.9% saline. First 4 rows of PSTHs, from left to right: responses to increasing doses of the indicated chemical (3 left PSTHs) and to repeated application at 1 dose (3 right PSTHs). Row 5: responses to repeated ethanol. Row 6: responses to repeated capsaicin. Bottom row, from left to right: responses to physical stimuli, acidified phosphate buffer (pH 4), mustard oil, and repeated application of piperine. Bottom left inset: receptive field (hatching) on ipsilateral eye and face. Bottom right inset: recording site () on drawing of brain stem section.
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The order in which the various chemicals were presented did not appear to influence whether the unit responded to subsequent chemicals, with the exception of capsaicin. Thus the 8 units tested first with histamine responded to 75.9% of the additionally tested chemicals, 10 tested first with acid responded to 78.6%, 6 tested first with NaCl responded to 71.4%, and 1 tested first with nicotine responded to 100%, of additionally tested chemicals. The three units tested first with 5-HT responded to only 31.8% of the additionally tested chemicals; two of these units were also unresponsive to 5-HT. The one unit tested initially with capsaicin did not respond to any of five subsequently presented chemicals (Table 2).
Time Course of Chemically Evoked Responses
TONGUE UNITS.
To compare the temporal profile of unit responses to the application of different chemicals to the tongue, the initial response of each unit to each chemical at a suprathreshold concentration was selected. Responses to each chemical were averaged across units and are shown in Fig. 4 aligned with stimulus application (
). The averaged responses increased significantly within 1-5 s after application of each chemical except ethanol (Fig. 4), although there were apparent differences in the time courses. Responses to histamine and 5-HT achieved a maximal firing rate most rapidly, whereas responses to nicotine, NaCl, ethanol, acid (pH), capsaicin, and mustard oil built up more slowly to the peak response (Table 3). The response to 5-HT was biphasic because some units gave a rapid and brief response, whereas others gave a slower and more prolonged response (Fig. 4). Peak firing rates were approximately equivalent for each chemical (Table 3). After the peak firing rate was achieved, responses declined during the 60-s period (Fig. 4) but remained significantly elevated throughout for all chemicals except histamine, nicotine, and mustard oil. The mean response evoked by histamine declined most quickly and was no longer significantly elevated 10 s after application. The mean nicotine-evoked response was no longer significantly elevated after 34 s, and the mean response to mustard oil was no longer significantly elevated after 30 s.
CORNEAL-CONJUNCTIVAL UNITS.
Averaged responses of these units to eight different chemicals are shown in Fig. 5. The brief initial peaks coincident with chemical application (Fig. 5, ) represent the mechanical response component that was apparent in approximately one-half of the units. For each chemical, there was an increase in the mean firing rate after the mechanical response component. Peak firing rates were reached most quickly (Table 3), and declined most rapidly (Fig. 5), with histamine, nicotine, and ethanol. The peak response took longer to build up with NaCl, acid, and mustard oil (Table 3) and declined more slowly; firing rates were still significantly elevated 60 s later (Fig. 5). Response profiles for capsaicin and acid (pH) were both characterized by a steady elevation in firing rate that persisted throughout the 60-s stimulus period (Fig. 5). Highest, and approximately equivalent, mean maximal firing rates were achieved after nicotine, ethanol, NaCl, and mustard oil, whereas 5-HT and piperine were least effective (Table 3; Fig. 5).
Dose-response relationship and tachyphylaxis
TONGUE UNITS.
Histamine. Most units responded to application of histamine to the tongue only at higher concentrations (1, 10%). Figure 6A shows individual dose-response curves for 20 units (thin lines) as well as the mean dose-response curve (thick line with error bars). The spontaneous activity (SA) level for each unit also is plotted. The treatment (dose) effect was significant (F = 27.09, P = 0.0001). Posthoc comparison revealed that the response to 10% histamine was significantly greater than that to 1% (Fig. 6A, *).
Figure 6B plots individual unit responses across histamine application trials repeated at 5-min interstimulus intervals. A few units exhibited declining or increasing responses, whereas most were fairly constant with no significant change across trials.
Nicotine. Figure 6C plots responses versus nicotine concentration for 16 units. Overall, responses increased significantly with dose of nicotine (F = 31.24, P = 0.0001), with responses to 1 and 10% significantly greater compared with lower doses. Figure 6D plots responses versus nicotine application trial. Mean responses significantly declined across trials (F = 41.7, P = 0.0001), with the mean response to trial 2 significantly lower compared with trial 1, and the mean response to trial 3 significantly lower compared with trial 2. Responses to trials 2 and 3 were 74 and 59.2% of trial 1, respectively.
NaCl. Unit responses increased significantly as a function of NaCl concentration (Fig. 6E; F = 13.79, P = 0.0001), with the mean response at 5 M being significantly larger compared with lower concentrations. In Fig. 6F, it can be seen that some units' responses declined over repeated trials of NaCl application, whereas many remained constant. On average, however, the declines at trial 2 and trial 3 (84.7 and 77.7% of trial 1, respectively) were not significant.
Ethanol. Unit responses increased significantly as a function of ethanol (EtOH) concentration (Fig. 6G; F = 23.79, P = 0.0001) with the response at 25% ethanol significantly larger compared with lower concentrations. Figure 6H shows that on average, responses to ethanol application did not change significantly across trials (mean response, trial 3 = 83% of trial 1).
Responses evoked by ethanol cannot be attributed solely to evaporative cooling of the tongue because four units that responded to ethanol did not respond to cooling the tongue and one unit unresponsive to ethanol did respond to tongue cooling (Table 1). Cooling may have contributed to the response evoked by ethanol in the seven units that responded to both.
5-HT. Mean responses increased significantly as a function of 5-HT concentration (Fig. 6I; F = 8.44, P = 0.0057) with the response to 5% significantly larger than the spontaneous rate. Figure 6J plots responses across application trials. The mean response to the second trial was significantly lower (62.5%) compared with the first trial. The mean response to the third trial was not significantly different compared with the first trial.
Acid (pH). Although the mean dose-response curve (Fig. 6K) was fairly flat, there was a significant effect (F = 21, P = 0.0001) with the mean response at pH 1 being significantly larger compared with responses at higher pH values. Figure 6L shows that responses of most units were fairly consistent across trials, with no significant change in mean responses.
Capsaicin. The determination of dose-response relationships with capsaicin proved to be difficult because the first effective dose of capsaicin often resulted in an apparent desensitization. Although many units exhibited an increased response to a suprathreshold versus subthreshold dose of capsaicin (e.g., 0.001 vs. 0.01% or 0.01 vs. 0.1%; Fig. 6M), subsequent responses to even higher concentrations of capsaicin were usually smaller than the initial response. This is reflected in the mean dose-response curve in Fig. 6M, which nonetheless demonstrated a significant dose effect (F = 16.26, P = 0.0001) in which the response to 0.01% capsaicin was significantly larger compared with the lower dose. Figure 6N shows that responses to subsequent applications of capsaicin declined significantly (F = 30.7, P = 0.0001). The response to trial 2 was 46.4% of the initial response.
After application of a suprathreshold concentration of capsaicin, units often became unresponsive to other chemicals as well. Thus 6 of 12 units were unresponsive to mustard oil, 4 of 9 were unresponsive to piperine, 2 of 2 were unresponsive to acid at pH 1, 2 of 3 were unresponsive to histamine, and 1 of 1 was unresponsive to nicotine after prior application of capsaicin. One unit that did not respond to capsaicin had received prior mustard oil.
A marked increase in spontaneous activity after initial application of capsaicin was observed in most units tested and is illustrated in Fig. 7A. The left PSTH shows a unit's low spontaneous firing level before and after initial application of capsaicin. The two middle PSTHs in Fig. 7A show dramatic increases in spontaneous firing 8 and 15 min after application of capsaicin. The spontaneous level had decreased but had not reattained the precapsaicin level, after 1 h (Fig. 7A, right PSTH). Typically, spontaneous firing waxed and waned in frequency as evident in Fig. 7A. The spontaneous firing rate at various times following the initial application of capsaicin was averaged in five units (Fig. 7B). It was significantly higher 4-6 min after the initial application of capsaicin (P = 0.0224, paired t-test) compared with the precapsaicin level and declined during the next hour (Fig. 7B).
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| FIG. 7.
Increase in spontaneous firing after initial capsaicin application. A: PSTHs show an individual unit's initial low firing rate immediately after application of capsaicin (left PSTH), followed by a progressive increase in spontaneous firing 8 and 15 min after capsaicin (middle PSTHs), which declined after 1 h (right PSTH). Note also waxing and waning of spontaneous rate. B: graph plots mean spontaneous activity (60 s epochs) before (pre), immediately after application of capsaicin (cap), and at progressively later time periods after initial capsaicin. * Significantly larger than precap response (P < 0.05, paired t-test).
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Piperine. Figure 6O shows that responses of the three units tested increased from 0.1 to 1% piperine although the sample size was insufficient for statistical testing. Mean responses significantly decreased across trials (Fig. 6P; F = 99.7, P = 0.0001) with the mean response to the second application being significantly lower (46%) compared with the initial response.
Mustard oil. Figure 6Q shows that responses tended to increase with mustard oil concentration in four units tested, and Fig. 6R shows that responses declined over repeated application trials in three units but first increased in a fourth unit.
Carbonated water. Each of four units responded to application of fresh carbonated water to the tongue. An example is shown in Fig. 8A. The unit did not respond to application of phosphate buffer at pH 6 (Fig. 8A, right PSTH), indicating that excitation by the carbonated water (pH = 6.1) was not due solely to pH. Figure 8B shows that three units gave fairly reproducible responses while responses of one declined over repeated application trials.
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| FIG. 8.
Response to carbonated water. A: response of caudalis unit to carbonated water (left PSTH) and lack of response to phosphate buffer fixed at pH 6 (right PSTH). Inset: receptive field (black) on ipsilateral tongue and chin. B: graph as in Fig. 5 (right) plotting individual (thin lines) and mean (thick line) unit responses to repeated application of carbonated water (interstimulus interval: 5 min).
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CORNEAL-CONJUNCTIVAL UNITS.
Histamine. Figure 9A shows dose-response curves for responses of corneal-conjunctival units to histamine. The dose effect was significant (F = 13.2, P = 0.0001), with the mean response to 10% histamine being significantly larger compared with lower concentrations. Figure 9B shows responses to repeated trials of histamine application at one supramaximal concentration (10%). The overall effect was significant (F = 41.8, P = 0.0001) with the mean response to the second application trial being significantly smaller compared with the first and third trials.
Nicotine. Mean responses increased significantly with nicotine dose (Fig. 9C; F = 36.8, P = 0.0001); the mean response to 10% was significantly greater than to 1%, and the mean response to 1% was greater than to 0.1%. Mean responses declined significantly across repeated trials of nicotine (Fig. 9D; F = 15.9, P = 0.0001), with the mean responses to trials 2 and 3 being significantly smaller compared with trial 1.
NaCl. Mean responses increased significantly with NaCl dose (Fig. 9E; F = 178, P = 0.0001) with the response to 5 M NaCl greater than to lower doses, and the response to 2.5 M NaCl greater than the spontaneous level. Responses to repeated application of NaCl did not change significantly across trials (Fig. 9F).
Ethanol. Mean responses increased significantly with increasing ethanol concentration (Fig. 9G; F = 7.32, P = 0.0006) with the response to 50% ethanol being significantly greater than the spontaneous level. Mean responses to repeated application of ethanol decreased significantly (Fig. 9H; F = 58.4, P = 0.0001) with the responses to the second and third trials being significantly lower compared with the first trial.
Of 10 units tested with both ethanol and cooling stimuli, 6 units were activated by both cooling and ethanol, 2 were excited by ethanol but not cooling, and 2 were excited by cooling but not ethanol. These data indicate that unit responses to ethanol were not due exclusively to evaporative cooling.
5-HT. Our sample of units was fairly unresponsive to 5-HT. Dose-response relationship was determined for only one unit (Fig. 9I), and responses of 2 of 4 units decreased with repeated 5-HT application (Fig. 9J). The sample size was too small for statistical analysis.
Acid (pH). There was considerable variability in unit responses to acidified buffer. For this reason, units were divided into groups giving maximal responses of either <200 (Fig. 9K) or >200 imp/60 s (Fig. 9L). Responses of most units tended to increase with decreasing pH, and the dose effect was significant for all units pooled (F = 5.5, P = 0.0012). For the group exhibiting lower firing rates (Fig. 9K), the mean response to acid at pH 1 was significantly larger compared with the spontaneous firing rate. However, a number of units that responded to acid at pH 3 or 4 gave smaller responses at lower pH levels (Fig. 9, K and L). When acidified buffer at a constant pH of 1 was applied repeatedly, successive responses declined significantly (Fig. 9M; F = 15.9, P = 0.0001) with the response to the third trial being significantly lower compared with the first trial.
Capsaicin. Mean responses significantly increased with capsaicin dose (Fig. 9N; F = 12.63, P = 0.0001) with the response to 0.1% capsaicin being significantly larger than to lower doses. Figure 9O shows that the responses of all six units tested decreased from the first to second application of capsaicin and this effect was significant (F = 31.38, P = 0.0001).
An increase in the spontaneous firing rate was noted in five units after initial application of capsaicin to the eye, but this was not systematically investigated further. We also did not systematically investigate possible cross-desensitization effects of capsaicin on responses evoked by subsequent application of other chemicals to the eye. After application of capsaicin, 9 of 15 units responded to mustard oil and 7 of 11 responded to piperine. However, because these latter chemicals were only rarely tested without prior application of capsaicin, we cannot be certain if the incidence of responsiveness to mustard oil and piperine was reduced by prior capsaicin.
Mustard oil. Mean responses increased significantly as a function of mustard oil concentration (Fig. 9P; F = 9, P = 0.006), with the mean response to 50% mustard oil being significantly larger compared with the spontaneous level. In Fig. 9Q, responses of 8 of 10 units decreased across trials of repeated mustard oil application although the overall change was not significant.
Piperine.
Figure 9R shows that the responses of the three units tested increased with concentration of piperine. The responses of all three units decreased over repeated trials of piperine application although the responses of two first increased from trial 1 to trial 2 (Fig. 9S).
Antagonists
TONGUE UNITS.
Many caudalis units responded to different classes of irritant chemicals. We wished to determine if the response to a particular chemical was mediated by a transduction mechanism specific to that chemical. Although transduction mechanisms for some of the presently tested chemicals are not known, responses to histamine and nicotine presumably are mediated by H1 and neuronal nicotinic receptors, respectively. We therefore investigated if an H1 antagonist, ceterizine, or the nicotinic ganglionic blocker, mecamylamine, could reduce or abolish the excitatory effect of histamine or nicotine, respectively.
Figure 10A shows responses of a dorsomedial caudalis unit to application of histamine to the tongue, before (Fig. 10A, top left) and 30 s after topical application of cetirizine to the tongue (Fig. 10A, top middle). Immediately after cetirizine the response to histamine was markedly reduced. Five minutes later the histamine response had fully recovered (Fig. 10A, top right). Figure 10A, middle, shows that identical application of cetirizine had no effect on the same unit's responses to nicotine. As shown in Fig. 10A, bottom, the response to nicotine (left) was reduced markedly after application of mecamylamine to the tongue (middle); the nicotine response had recovered partly 1 h later (right). Figure 10B shows data from another unit in which mecamylamine markedly reduced the response evoked by application of nicotine to the tongue (top middle), with little recovery 20 min later (top right), whereas mecamylamine did not reduce the same unit's response to histamine (Fig. 10B, bottom). Application of cetirizine or mecamylamine alone did not evoke responses in any units.
Responses to application of histamine to the tongue were significantly attenuated (to 47.5%; P = 0.033, paired t-test) after cetirizine (1%) in each of 10 units tested. Responses subsequently recovered to the control level within 5 min in 9 of 10 units, whereas in 1 unit the histamine-evoked response was suppressed only after a delay of 15 min after application of cetirizine. A lower dose of ceterizine (0.1%) also reduced responses to histamine in 2 of 3 units tested.
Responses to application of nicotine to the tongue were also significantly attenuated (to 33%, P < 0.05, paired t-test) 30 s after mecamylamine in four units. Partial recovery of the response to nicotine was only observed in 2 of 4 units during the next 30-60 min.
CORNEAL-CONJUNCTIVAL UNITS.
The mean response evoked by application of nicotine to the eye was significantly attenuated (to 65%; P = 0.004, paired t-test) immediately after application of mecamylamine (0.1%) in four units with full recovery 5 min later. An example is shown in Fig. 11. The histamine antagonist was not tested with corneal-conjunctival units because they exhibited a lower incidence of responsiveness to histamine (Table 2).
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| FIG. 11.
Mecamylamine antagonism of unit response to application of nicotine to the cornea-conjunctiva. PSTHs (binwidth: 1 s) show, from left to right, responses to application of nicotine (1%) to the cornea-conjunctiva, the response when nicotine was applied 30 s after corneal application of mecamylamine (0.1%), and the response to nicotine 5 min later.
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DISCUSSION |
These results have identified a population of WDR and nociceptive-specific neurons in the superficial laminae of the dorsomedial aspect of trigeminal subnucleus caudalis that was activated by irritant chemical stimulation of the tongue, as well as a second population of WDR neurons in ventrolateral caudalis that was also activated by irritant chemical stimulation of the cornea-conjunctiva. A salient finding is that most caudalis neurons responded to application of a broad spectrum of irritant chemicals to the tongue or ocular mucosa (Tables 1 and 2). Furthermore, responses to histamine and nicotine were reduced or prevented by prior application of H1 or nicotinic antagonists, respectively. Therefore our data indicate that a substantial fraction of trigeminal caudalis units are activated by multiple irritant chemicals at least partly via
Abstract
Introduction
Abstract
