Taste bud distribution and innervation on the palate of the rat

Chemical Senses Volume 7 Number 1 1982 Taste bud distribution and innervation on the palate of the rat Inglis J.Miller,Jr. and Kevin M.Spangler Department of Anatomy, Wake Forest University, Bowman Gray School of Medicine, Winston-Salem, NC 27103, USA (Received October 1981; revised June 1982; accepted June 1982) Abstract. The functional properties of taste buds on the palate have not been investigated in laboratory mammals due to limited information about their spatial distribution and innervation. Three regions of the rat's palate contain a mean total of 227 taste buds. The nasoincisor ducts (NID) are located on the incisal papilla at the first antemolar ruga and contain a mean of 66 taste buds (29% of total) divided between the two ducts. About four taste buds (1.8% of total) on the NID survive bilateral transection of the greater superficial petrosal nerve (GSP). At the boundary between the hard and soft palate is a narrow strip of taste buds termed the 'Geschmacksstreifen' (OS). This bilateral structure contains a mean of 69 taste buds (30% of total), all of which degenerate with transection of the GSP. The posterior palatine field (PPF) of the soft palate contains a mean of 92 taste buds (41% of total) clustered along the midline from the GS to the nasopharynx. A mean of 29.9 (13.2% of total) taste buds on the PPF survive GSP transection. The distribution of the GSP from both sides overlaps bilaterally to a high degree. It is concluded that 85% of the palatal taste buds in the rat are innervated by the GSP division of the facial nerve, while the remaining 15% are probably innervated by glossopharyngeal fibers which reach the palate by way of the pterygopalatine artery. Introduction Palatal taste buds have been identified in several species of experimental animals and in humans. A survey among species was made by Kaplick (1953) who reported taste buds on the soft palate of the rat, rabbit, hamster, guinea pig and hedgehog. He found no taste buds on the palates of swine, cattle, sheep, dog, cat and man. Some of Kaplick's observations were obtained from stained, wholemounted specimens, while others came from sectioned tissue. His negative results, especially from humans and sheep, have been questioned in the light of contemporary observations. The taste buds on the rat's palate were described in three regions: in the nasoincisor or nasopalatine ducts, at the boundary of the hard and soft palates in a structure referred to as the 'Geschmacksstreifen' (GS) or taste stripe, and along the surface of the soft palate. Taste receptors have been identified in limited numbers on the soft palates of human adults (Nilsson, 1979), but they were found in abundance in the case of one human newborn (Lalonde and Eglitis, 1961). Nilsson obtained disparate results from two sources. Autopsy specimens comprising several square centimeters were sectioned serially in paraffin; every third section was examined; but no taste buds were observed. Biopsy samples consisting of 11 mm 2 circular pieces of tissue were embedded in Epon and sectioned at 1 /*m. Taste buds were observed in the biopsy samples. Although taste buds are found on human soft palates, an estimate of their number and spatial distribution would be questionable based upon accumulated evidence. Innervation of the rat's palate is derived from three pairs of palatine nerves IRL Press Limited, Oxford, England. 0379-864X/82/0701-0099$2.00/0 99

I.J.Miller.Jr., and K.M.Spangler which contain trigeminal axons as well as facial sensory fibers from the greater superficial petrosal nerve (GSP) (Miller, Gomez and Lubarsky, 1978). Cleaton- Jones (1976) reported that bilateral transection of the GSP produced degeneration of about two-thirds of the taste buds on the soft palate of the rat. Functional studies of palatal taste buds have been limited in laboratory animals (see review, Miller, 1977) because of the dearth of information about the distribution of the receptors and their innervation. The current study was undertaken to identify the number and spatial array of taste buds in each of the palatal regions as an antecedent to the development of stimulation techniques for functional studies. Methods Animals The subjects were adult, male Sprague-Dawley rats which weighed 200-250 g at the beginning of the experiment. All were anesthetized by i.p. injection of sodium pentobarbital which averaged - 50 mg/kg of body weight. Six rats served as controls, and in nine rats the GSP was transected. Nerve transection The GSP was transected bilaterally in eight rats and unilaterally in one rat. An incision was made medial to the pinna on the parietal aspect of the head above the ear. Blunt dissection was used to isolate the external auditory meatus, and the auditory canal was transected to expose the bony portion of the meatus. Small rongeurs were used to enlarge the meatus followed by removal of the tympanum and ossicles. Retraction of the tensor tympani muscle and removal of a thin layer of temporal bone exposed the GSP distal to the geniculate ganglion. The nerve was avulsed with jeweler's forceps, and the skin was closed with superficial sutures. The animals were reanesthetized 20 22 days following transection and sacrified by intraventricular perfusion of Bouin's fixative. At the time of sacrifice, the animals weighed 300-350 g. Autopsy revealed that seven of eight bilateral transections yielded successful surgical preparations. In one animal the left side was transected while the right side was incompletely transected. The unilateral transection resulted in a successful operation. Tissue preparation Palates were removed from the transverse terminal ridge caudally to the nasopharyngeal orifice by cutting with a scalpel along the medial aspect of the pterygoid plate. The central portion of the first antemolar ruga, containing the nasoincisor ducts (NIDs), was excised. Tissue specimens were stored for 24 h in fixative, dehydrated in alcohol, cleared in toluene or xylene and embedded in Paraplast. NIDs were oriented for sectioning in the transverse plane and soft palates were oriented in the parasagittal plane. Serial sections were cut at a thickness of 12 15/im and mounted on glass microscope slides. Slides were stained with routine hematoxylin and eosin or FitzGerald's (1964) silver technique. Complete serial sections were obtained, and each section was examined. The 100

Taste bud distribution and innervation on the palate of the rat location of taste buds was plotted on a chart by measuring the distance from the taste bud to the GS or the nasopharyngeal orifice. The section containing the taste pore was noted on the chart, and the location of each taste bud was established, independently, by two observers. Structural features of the palate are named according to the terminology of Kutuzov and Sicher (1952) and Kaplick (1953). Results Spatial distribution of taste buds on the palate Taste buds were found in three regions of the palate which include: the NIDs, the GS and the posterior palatine field (PPF). The three regions are shown in Figure 1 from a midsagittal section of the entire head of a 22-day-old rat. Bilateral NIDs ducts connect the nasal cavity with the oral cavity and open onto the palatal surface of the incisal papilla. This papilla is fused with the first antemolar ruga (Figure 1B), and one duct emerges from each lateral surface with a crescent-shaped opening which is located 1 mm from the midline. Taste buds were found on the medial walls of the ducts within 100 ^m of the palatal surface (Figure 1 A), but a few were located on the lateral surface of the ruga. Six normal rats had a mean total of 66.3 ±7.4 (s.d.) taste buds on the two ducts and adjacent ruga with a range of 14 41 on each side. Taste buds were about equally divided between the two sides: left 36 ±5; right 31 ±10. At the boundary between the hard and soft palate is a region of thickened epithelium on the hard palate called the transverse terminal ridge (TR), and caudal to it on the soft palate is a bilaterally paired structure described by Kaplick as the GS (Figures 1C and ID). Each lateral half of the GS extends 2-3 mm in the transverse plane and contains taste buds which can be seen in groups of 2-3 on the sagittal axis (1C). The two halves of the GS are separated by 0.5 mm in the midline. An average of 68.8 ±8.5 (s.d., n = 6) total taste buds was found on the apical surface of the GS with a range of 22-44 on each half. The caudal portion of the soft palate extends -1 cm in length from the GS to the nasopharynx, and is supported on each side by the pterygoid plates. Taste buds are found singly or in groups of up to three on slight eminences (Figure IE) which are clustered along the midline of the PPF, and there is usually no distinct separation of taste buds on the lateral halves of the soft palate. Mucous glands (Figure IF) empty onto the palatal surface via ducts that emerge throughout its length (Figure IF, asterisk). An average of 92.2 ± 18.2 (s.d.) taste buds was found on the PPF with a range of 73 117 among six control rats. The spatial distribution of taste buds on the soft palates of normal rats is illustrated in Figure 2. The total number of taste buds on the soft palate (GS + PPF) was 161 ±22 (n = 6) with a range of 134 194. Including taste buds on the NIDs, the total of taste buds on the entire palate was 227 ±21 (n = 6) with a range of 209-258. Taste bud distribution following GSP transection Bilateral transection of the GSP in seven rats produced a reduction in the total number of palatine taste buds by 85 % after 20-22 days of post-surgical survival. 101

l.j.miller,jr., and K.M.Spangler Fig. 1. Regions of taste buds on the rat's palate. Center panel: sagittal section through the entire head of a 22 day old rat. Incisal papilla containing NID; transverse terminal ridge (TR); GS and PPF. The entire length of the panel represents 32 mm. Panel A: transverse section of NID with taste buds indicated, lumen of duct: white arrows. Entire panel width: 0.35 mm. Panel B: location of incisal papilla and first antemolar ruga. Panel width: 40 mm. Panel C: Sagittal section through GS showing taste buds, TR to right side of section. Panel C width: 0.2 mm. Panel D: gross aspects of GS and TR showing one lateral half of palate. Panel D width 3.0 mm. Panel E: taste buds on PPF. Panel E width: 0.25 mm. Panel F: PPF containing mucous glands and opening of duct (asterisk) onto palatal surface. Panel F width: 1.25 mm. Figure 3 shows the mean number of taste buds in the NID, GS and PPF of the control group in comparison to the number of taste buds found after GSP transection. The greatest reduction occurred on the GS which contained no taste 102

Taste bud distribution and innervation on the palate of the rat Fig. 2. Taste bud distribution on the soft palate of five control rats. buds after transection of the GSP. The NIDs contained a mean toal (±s.d.) of 4.1 ±3.8 {n = 7.) taste buds (range 0-10) compared with a mean total of 63.7 ±7.4 taste buds on the control animals. The PPF contained a mean total of 29.9 ± 15 («= 7) taste buds (range 2 54) compared with a mean total of 92 ±17 («= 6) taste buds on the PPF of control animals. Palates of animals with GSP transection contained a mean total of 34 ± 17 taste buds which remained, compared with a mean total of 227.3 ±21 (n = 6) taste buds in controls. The spatial distribution of surviving taste buds on the soft palate following GSP transection is illustrated in Figure 4 in comparison with a normal palate. Note the total absence of taste buds on the GS of lesioned animals. Transection of the GSP reduced the number of taste buds on the PPF to 32.5% of controls, but the diminution was greatest in the rostral portion of the PPF. On the rostral one-third of the PPF, 6.5% of the taste buds survived GSP transection. The middle one-third of the PPF contained 32.3% of the control number of taste buds after the GSP was cut; while on the caudal one-third of the PPF 61.6% of the taste buds survived GSP transection. Unilateral transection of the GSP produced a result which was intermediate between the normal distribution of taste buds on the soft palate and the distribution which resulted from bilateral transection. In one animal with transection of the left GSP, eight taste buds remained on the left NID and 48 taste buds were found on the right NID. Nine taste buds were located on the left GS, while the right GS contained 33 taste buds. The posterior palatine field retained 102 taste buds after transection of the left GSP. Thus, unilateral transection of the GSP reduced the total number of palatine taste buds in one rat by only 12% compared with controls. The total number of taste buds on the GS was reduced to 39% of controls, while the total number of taste buds in the NID was diminished by 103

I.J.Miller.Jr., and K.M.Spangler 100 N=6 CONTROLS EH N=7 LESION ^ M^1 NASOINCISOR DUCTS GESCHMACKS- STREIFEN POSTERIOR PALATINE FIELD Fig. 3. Mean taste bud distribution among three regions of the palate for control rats (unfilled bars) and following GSP transection (cross-hatched bars). ~15%. In another animal with complete transection of the left GSP and incomplete transection of the right GSP, there remained a total of 76 taste buds with a 77% reduction in the GS, a reduction of 71% on the NID and a reduction of 55% on the PPF. Discussion The current report of three groups of taste buds on the palate corroborates the findings of Kaplick (1953). Several investigators have described taste buds on the palate of the rat (Kutuzov and Sicher, 1952; Cleaton-Jones, 1971, 1976; Smith and Calhoun,- 1972; Mistretta, 1972) and in the nasopalatine ducts (Kolmer, 1927). The objective of the current study was to provide sufficient definition of the spatial distribution of palatal taste buds and their innervation in order to permit discrete stimulation with chemicals for physiological studies. A mean total of 227 ±21 (s.d., n = 6) palatal taste buds are distributed among the three regions as follows: NID (29%), GS (30%) and PPF (41%). This total represents ~ 17% of the entire oropharyngeal complement of taste buds (Miller, 1977) which is a larger proportion than had been recognized (Pfaffmann, 1952). Following bilateral transection of the GSP, 34 ±17 (s.d., «= 7) palatal taste 104

Taste bud distribution and innervation on the palate of the rat Fig. 4. Taste bud distribution on four rat palates following GSP transection (B-E) compared with one normal (A). The orientation is the same as Figure 2. buds survived. This represents 15% of the total (227) of which 1.8% were located on the NID and 13.2% were found on the PPF. All of the taste buds on the GS degenerate when the GSP is cut. About one-third of the taste buds on the PPF survive GSP transection with a decreasing gradient of degeneration from the rostral to the caudal region. Unilateral transection of the GSP by Cleaton-Jones (1976) produced degeneration of only 17% of the taste buds on the soft palate. The limited results of unilateral transection in the present study corroborate the conclusion of Cleaton-Jones that the palatine nerves overlap bilaterally in their distribution to the palate. It seems reasonable to conclude that the 85% of palatal taste buds which degenerated following bilateral GSP transection were innervated by the facial nerve. A mean total of 29.9 ±17 (s.d., n = 7) surviving taste buds was observed on the soft palate after bilateral GSP transection in this study, compared with a mean survival of 51.7 ±4.8 (s.e., n = 3) taste buds after 14 days in the report by Cleaton-Jones (1976). Two features differ among the methods used in the two studies. Cleaton-Jones utilized survival times of 7, 14 and 28 days compared with 20 22 days in the present study. He found no substantial changes in the interval from 14 to 28 days of degeneration, so this difference seems to be inconsequential. The major difference between the two studies was in the location of the GSP transection. The site of the lesion in the current study was with an approach through the middle ear -2 mm distal to the geniculate ganglion. The apparent 105

I.J.Miller,Jr., and K.M.Spangler site of the lesion by Cleaton-Jones was between the Eustachian tube and the medial pterygoid plate which is -5 10 mm distal to the site of the lesion in this study. Between the two lesion sites the GSP intersects with the deep petrosal nerve from the carotid plexus (Zacharias, 1941) which follows the internal carotid artery. The disparity in surviving taste bud numbers between the two experiments may have resulted from facial sensory axons which depart from the geniculate ganglion with the GSP but deviate from the vidian nerve proximal to the lesion site of Cleaton-Jones to reach the palate by following the vascular and autonomic nerve supply. It is apparent from the current study and from the report of Cleaton-Jones that a source of innervation other than the GSP innervates taste buds on the soft palate of the rat. The distribution of surviving taste buds following bilateral GSP transection shows an absence on the GS and an increasing gradient of survival toward the nasopharynx (Figure4). This observation suggests that the 15% of the total palatine taste buds which survive GSP transection may be innervated by nerves which supply the soft palate from the caudal region. Cleaton-Jones reported that bilateral transection of the glossopharyngeal nerves in combination with bilateral GSP transection failed to decrease the number of surviving taste buds compared to the number surviving GSP transection alone. In four cases (two unilateral, two bilateral) we failed to detect any palatal taste bud degeneration following glossopharyngeal nerve transection as the nerve exits the posterior lacerated foramen (unpublished observations). Taste buds on the superior aspect of the nasopharynx survive bilateral GSP transection (unpublished observations), but the source of their innervation remains unestablished. Electrophysiological responses were reported from mechanoreceptors in the epipharynx of the rat by Nail et al. (1969) that were innervated by the pharyngeal branch of the glossopharyngeal nerve. Lawson (1980) found branches of the prelingual glossopharyngeal nerve in the rat which innervated the caudal portion of the soft palate and pharynx in a manner homologous to the tonsillar branches of the human glossopharyngeal nerve. Transection of the glossopharyngeal trunk as it emerges from the posterior lacerated foramen should degenerate both of these divisions, so it would seem that they are poor candidates for the supply of taste buds which survive GSP transection. In sectioned material from human embryos, Kanagasuntheram et al. (1969) described nerve fibers which arose from the inferior ganglion of the glossopharyngeal nerve or from the proximal portion of the nerve trunk to join the internal carotid nerve plexus of sympathetic fibers. As pointed out above, this plexus joins the GSP as the deep petrosal nerve; while some axons follow blood vessels to their target organs. Since the pterygopalatine artery, which supplies the palate, arises from the internal carotid artery in the rat (Green, 1959), it seems plausible that sensory axons from the inferior ganglion of the glossopharyngeal nerve follow the carotid artery to its union with the pterygopalatine artery and, thence, to the palate. As suggested above, some axons from the GSP may reach the palate by the same route. This course for glossopharyngeal fibers would survive transection of the nerve trunk as well as GSP transection, and it probably ac- 106

Taste bud distribution and innervation on the palate of the rat Fig. 5. Diagram of oropharyngeal taste bud distribution by region. A, fungiform papillae; B, circumvallate papilla; C, foliate papillae; D, epiglottis; E, nasoincisor ducts; F, GS; G, posterior palatine field. counts for the 15% of palatal taste buds which persist after transection in the current study. Although little is known about the functional properties of palatal taste buds, their spatial distribution can be examined in relation to the other taste bud populations in the rat. Figure 5 shows the major locations of taste buds in the rat from a prior report (Miller, 1977). Taste buds in the nasoincisor ducts (E) are apposed to the high density of fungiform taste buds on the tip of the tongue (A). Taste buds of the GS (F) are adjacent to the circumvallate papilla (B) at the midline and the foliate papillae (C) on both lateral surfaces of the tongue. Abundant taste buds on and around the epiglottis are apposed to the taste buds of the posterior palatine field (C). During swallowing, ingesta is forced between the tongue and palate which must result in simultaneous stimulation of lingual and palatal taste buds. In the rat there are a few additional taste buds in the orifice of the submandibular duct beneath the tip of the tongue, on the buccal wall lateral to the foliate papillae, on the superior aspect of the nasopharynx and in the rostral end of the esophagus (personal unpublished observations). Of an approximate total of 1265 taste buds in the rat (Miller, 1977), the palate including the NIDs contain -17%, the fungiform papillae -15%, the foliates -36%, the circumvallate -28% and the epiglottis contains -4%. Based upon their spatial distribution, it seems reasonable that palatal taste buds function in temporal register with taste bud populations in contiguous regions of the tongue. Acknowledgements The authors are indebted to Henry Moss and Janice Collins for technical assistance. Len Porcelli collected the majority of observations from control animals. Preliminary reports of some of these observations have been presented 107

I.J.Miller,Jr., and K.M.Spangler at the 89th Session of the American Association of Anatomists (Anat. Rec, 184, 480, 1976) and the International Symposium on Food Intake and Chemical Senses, Fukuoka City, Japan, 1976 (see ref. Miller, 1977). This work was supported, in part, by NIH Grant DE 05259 from NIDR. References Cleaton-Jones.P.: 1971, 'Histological observations in the soft palate of the albino rat', J. Anat., 110, 39-47. Cleaton-Jones.P.: 1976, 'A denervation study of taste buds in the soft palate of the albino rat', Arch. Oral Biol., 21, 79-82. FitzGerald.M.J.T.: 1964, 'The double-impregnation silver technique for nerve fibers in paraffin sections', J. Micro. Sci., 105, 359-361. Greene,E.C: 1959, 'Anatomy of the Rat', Hafner Publishing Co., NY, pp. 370. Kanagasuntheram.R., Wong.W.C. and Chan.H.K.: 1969, 'Some observations of the innervation of the human nasopharynx', J. Anat., 104, 361-376. Kaplick.M.: 1953, 'Uber Vorkommen, Verteilung und Histologische Beziehungen der Geschmacksknospen am Mundach einiger Sauger, besonders der Nagetier', Zeit. Zell., 38, 571-590. Kolmer.W.: 1927, 'Uber das Vorkommen von Geschmacksknospen im Ductus Naso-palatinus der Ratte', Anat. Anz., 63, 248-251. Kutuzov,H. and Sicher.H.: 1952, 'Anatomy and function of the palate in the white rat', Anat. Rec, 114, 67-84. Lalonde.E. and Eglitis.J.: 1961, 'Number and distribution of taste buds on the epiglottis, pharynx, larynx, soft palate and uvula in a human newborn', Anat. Rec, 140, 91-95. Lawson.J.W.: 1980, 'The lingual and pharyngeal distribution of the glossopharyngeal nerve in the rat', Unpublished Masters Thesis, Wake Forest University, Winston-Salem, North Carolina. Miller.I.J.: 1977, 'Gustatory eeceptors of the palate', in Katsuki et a/., (eds.), Food Intake and Chemical Senses, University of Tokyo Press, Tokyo, pp. 173-185. Miller.I.J., Gomez.M.M. and Lubarsky.E.H.: 1978, 'Distribution of the facial nerve to taste receptors in the rat', Chem. Senses Flavor, 3, 397-411. Mistretta.C: 1972, 'Topological and histological study of developing rat tongue, palate and taste bud', in Bosma.J. (ed.), Oral Sensation and Perception, C.C.Thomas, Springfield, pp. 163-187. Nail.B.S., Sterling.G.M. and Widdicombe.J.G.: 1969, 'Epipharyngeal receptors responding to mechanical stimulation', J. Physiol., 204, 91-98. Nilsson.B.: 1979, 'The occurrence of taste buds in the palate of human adults as evidenced by light microscopy', Acta Odont. Scand., 37, 253-258. Pfaffmann.C: 1952, 'Taste preference and aversion following lingual denervation', J. Comp. Physiol. Psychol., 45, 393-400. Zacharias.L.R.: 1941, 'Further studies in naso-genital relationship anatomical studies of the perihypophyseal region in the rat', J. Comp. Neurol., 74, 421-446. 108