- Email: [email protected]
The effect of neonatal capsaicin treatment on gustatory behavior in the albino rat
Physiology&Behavior,Vol. 52, pp. 1037-1042, 1992
0031-9384/92 $5.00 + .00 Copyright© 1992PergamonPressLtd.
Printed in the USA.
The Effect of Neonatal Capsaicin Treatment on Gustatory Behavior in the Albino Rat JUDITH
R. G A N C H R O W , .1 Z E ' E V S E L T Z E R * A N D N I T Z A N BITCHACHO~"
*The Hebrew University-Itadassah Faculty of Dental Medicine Founded by the Alpha Omega Fraternity, Jerusalem, Israel and y'The Goldshlager Faculty of Dental Medicine, Tel Aviv University, Tel Aviv, Israel R e c e i v e d 12 M a r c h 1992 GANCHROW, J. R., Z. SELTZER AND N. BITCHACHO. The effect of neonatal capsaicin treatment on gustatorybehaviorin the albino rat. PHYSIOL BEHAV 52(6) 1037-1042, 1992.--Small-diameter fibers present in gustatory peripheral nerves have historically been suspected of relaying information about the bitter quality of a taste stimulus. Neonatally injected capsaicin irreversibly destroys a proportion of unmyelinated C- and some A~-fibers.Consummatory responses to increasing concentrations of quinine and other chemical solutions following neonatal capsaicin injection were compared to those of untreated and vehicleinjected control Sabra albino rats. Capsaicin-treated rats significantly increased their withdrawal thresholds to noxious, CO2 lasergenerated heat pulses verifyingtreatment effectiveness.Furthermore, neonatal capsaicin treatment diminished sensitivity to pungent capsaicin solutions in mature rats. However, there were no group differences in quinine intake, suggesting that the full array of unmyelinated fibers associated with taste buds is not essential for the transmission of bitter taste. Capsaicin-treated animals showed a significant reduction in intake of normally highly preferred sodium chloride and sucrose concentrations. These results were probably not due to loss of peripheral unmyelinated afferent fibers per se, but rather to secondary central changes. Capsaicin
Taste
Preference
Consummatory behavior
GUSTATORY nerves are comprised of both myelinated and unmyelinated fibers (9,27,32). Some hypotheses concerning quality coding have suggested different roles for fibers of different diameters [e.g., (2,12)]. For instance, based on size, conduction velocity and/or scarcity, it has been suggested that the smallest diameter fibers convey bitter information (6,16,23,24). Furthermore, the population of afferent (unmyelinated) C-fibers in the glossopharyngeal nerve is markedly larger than that found in the chorda tympani nerve (32), possibly correlating with the larger proportion of glossopharyngeal fibers responding to quinine [e.g., (44)]. Capsaicin (trans-8-methyl-N-vanyllyl-6-nonanamide), the pungent substance in chili peppers, is known to produce rapid, irreversible destruction of many primary sensory C- and some A~-fibers when administered neonatally at a concentration of 50 mg/kg [e.g., (19,25,33,41,45)]. If the fine afferent fibers of the gustatory nerves were specifically labelled to carry qualitatively bitter information, one would expect neonatal capsaicin treatment to severely impair preference behavior, producing heightened thresholds and overall diminished responsivity to an increasing quinine concentration series. Some support for this hypothesis has been obtained following repeated capsaicin injections in adult rats (48), who subsequently showed decreased sensitivity only to higher concentrations of quinine in preference tests that included sodium chloride and citric acid as well. The purpose of this study was to examine the behavioral responses of adult rats to several concentrations of bitter, sweet,
Quinine
Sucrose
NaC1
Development
salty, and pungent stimuli, following a single neonatal capsaicin treatment. METHOD
Subjects Consummatory behavior was examined in 30 Sabra strain albino rats, divided into three groups. Members of each group were drawn from eight litters born within a week of each other. On postnatal day 2, 49 pups of either sex were injected subcutaneously with a 50 mg/kg dose of capsaicin dissolved in the vehicle [10% Tween 80, 10% ethyl alcohol in saline (24)] totalling 35 #l volume. In order to prevent leakage, the syringe needle was inserted at the nape of the neck and advanced subdermally such that injections were made at the lumbar region. At the same time, a comparable group of 14 littermate pups received the vehicle alone, and 12 pups served as untreated controls. All rats were weaned at 21 days and reared under similar group conditions and taste testing began on postnatal day 290. Following a lower survival rate among males treated neonatally with capsaicin, the remaining animals were divided into groups as follows: the capsaicin-treated group was comprised often females (average weight 229 g) and initially three surviving males (average weight 490 g) who died during the early phases of the experiment, and their data are, therefore, not included. The two control groups (vehicle and untreated) each included five females (average weights 227 and 232 g, respectively) and five males
Requests for reprints should be addressed to Dr. J. R. Ganchrow, Department of Oral Biology, The Hebrew University-Hadassah, Faculty of Dental Medicine, P.O.B. 1172, Jerusalem 91010, Israel. 1037
1038 (average weights 550 and 429 g, respectively). One female in the experimental group died during the last week of testing. The females receiving capsaicin gained an average of 64 g during the 62 days of testing, while the females of the vehicle group and the normal group gained an average of 64 and 56 g, respectively. The males of the latter two groups gained an average of 108 g and 82 g, respectively.
Procedures The experiment was carried out in a room under a 13 h-light (commencing at 0700 h), 11-h dark cycle. Room temperature was maintained at 27 + 1°C. Laboratory rat chow (Ambar Ltd. 931) was available ad lib throughout the experiment. Nociceptive reflex threshold test. Seven days before the start of the experiment, each animal was tested for withdrawal response thresholds to ultra short noxious heat pulses radiated from a CO2 laser (5 watts; 30-120 ms; 40-150 mcal) applied to the ear, nose, forepaw, hindpaw, and tail. The stimulated target (1.5 mm in diameter) was illuminated by a visible spot of red light from the He/Ne-aiming laser. Heat pulse intensity was gradually increased until the unrestrained animal responded by withdrawal which was recorded via pressure-transduced oscillographic traces and confirmed visually. Drinking preference test. A standard 2-bottle, 24-h testing procedure was utilized to monitor taste-guided fluid consumption. During preference testing, each animal was housed in a 24 × 35 × 12 cm wire mesh cage, suspended on a rack. Members of each group were randomly distributed into the bank of cages and their identity concealed such that data were recorded under blind conditions. Two 100 ml graduated glass cylinders fitted with rubber stoppers and L-shaped, drinking spouts were affixed to the front of each cage, about 4 cm from each lateral edge (separation distance = 16 cm), and 4 cm above the floor. A food tray occupied a position midway between the two drinking tubes. The tapering tip of each stainless steel drinking spout (inside tip diameter about 1.5 mm) inserted to about 2 cm inside the cage for fluid delivery. Each stimulus concentration was presented for choice against bidistilled water for a total of 48 h, 24 h on each side to balance out side preferences. Readings of the fluid volume contained in each bottle were taken to the nearest ml between 1115 and 1130 h each morning, and fluids were then either replenished or replaced, and their volumes again recorded. Solutions were prepared from reagent grade chemicals, and bidistilled water was the solvent. The test stimuli included sucrose (Suc) at 10, 30, and 300 mM; quinine hydrochloride (QHC1) at 1, 5, 10, 50, and 500 uM,' sodium chloride (NaCI) at 3, 10, 30, 100, 300, and 500 mM; and eapsaiein (Cap) dissolved in 100 ul ethanol (ETOH) per liter of water at 0.0016, 0.0023, 0.0033, and 0.016 mM. Since the capsaicin was by necessity dissolved in ethanol, 100 ul ETOH was likewise included in each liter of water presented for choice against the capsaicin solutions. This miniscule amount of ETOH in the distilled water was not detectable to the experimenters. Stimuli were presented in ascending concentrations in the above order with the addition of 50 # M QHC1 before and immediately following the capsaicin run, to observe whether drinking capsaicin in the days preceding quinine might diminish sensitivity to the latter. Three days of water available in both bottles interceded between each change in chemical category with the exception of the last quinine exposure, which occurred on the day immediately following the highest capsaicin concentration.
Data Anal.vsis Intake measures were standardized relative to the amount of fluid an individual rat would be likely to consume in 24 h from
GANCHROW, SELTZER AND BII'CttACHO one bottle, if both bottles contained water. Thus, mean 24-h solution intake was compared to the averaged daily one-bottle water intake for the 3 days immediately preceding and the 3 days immediately following any stimulus concentration series for each animal. Single bottle water intake is defined as half the total daily two-bottle water intake. Therefore, standard intake = [mean (2 day) solution intake/mean (6 day) single bottle water intake] × 20. This sets each rat's single bottle water intake at 20 and expresses all other intakes relative to this value. (The value 20 was arbitrarily chosen since the average 24-h water consumption in normal Sabra rats is around 40 ml, or theoretically 20 ml/bottle.) Thus, values greater than 20 would suggest a tendency toward preference, and less than 20, aversion. This transformation was utilized in an attempt to decrease the variability contributed by different fluid consumptions between individuals or between groups. It was deemed to be a more sensitive measure of taste reactivity than the usual within-trial two-bottle, 24-h percentage measure (percentage preference = [solution intake/ total intake] X 100%), where consumption from each bottle may be differentially affected both by taste and by physiological reactions, such as water regulation [see e.g., (13)] influenced by a variety of sources. Furthermore, this method avoids the problems of standardizing by animal weight [see (51)]. A new multiplication factor was obtained for each chemical category tested, to control for possible shifts in fluid consumption over the 2 months of testing, due to possible changes in seasonal climatic condition, animal aging, or other unknown factors. All group data are presented as means __ SEM. The rejection level for analyses of variance two-factor, mixed design, (7) and other statistical procedures was p < 0.05. Males and females were grouped together for statistical analysis except when examining responses to sucrose and NaC1, substances known to elicit sex differences in rat preference behavior [e.g., (4,14,28,50)]. RESULTS Capsaicin treatment profoundly decreased sensitivity to painful stimuli in the various extraoral body sites examined. The average withdrawal threshold of capsalcin-treated rats was 40°70 higher than that of the combined control groups, and this difference was significant both for the grouped data and for between group comparisons (e.g., for individual noxious stimulus sites the threshold ranges were t = 2.65-7.78, p -< 0.0005-0.01 for the capsalcin versus control group comparisons, and t = 2.21-5.07, p -< 0.0005-0.025 for the capsaicin versus vehicle group comparisons, one-tailed t-test). Intraoral sensitivity to irritants was also affected: neonatal capsaicin treatment diminished aversion to drinking capsaicin solutions in adult female rats. It may be seen in Fig. 1 that capsaicin-treated animals continued drinking capsaicin solutions up to 0.0033 m M in amounts similar to normal water drinking while the two control groups decreased their intake by about half in these concentration ranges. The group differences were significant, F(2, 27) = 5.45, p < 0.01, as were the concentration, F(3, 81) = 8.54, p < 0.01, and the interaction, F(6, 81) = 3.85, p < 0.0 l, effects. When males of the two control groups were compared to females of the two control groups, no significant differences were found, F(1, 18) = 0.52, p > 0.05, indicating that gender is not relevant to capsaicin drinking. These relationships and curve shapes were maintained when within-trial percentage preference was calculated. For example, at 0.0033 raM, percent preference values for capsaicin solutions were 52070, 32°70, and 1207ofor the capsaicin-treated, vehicle-treated, and untreated controls, respectively. In this kind of analysis, 50070 represents indifference and decreasing values signify increasing aversion. Thus, even at 0.0033 rnM the neonatally capsaicintreated group did not refrain from drinking capsaicin solutions.
CAPSAICIN TREATMENT AND TASTE BEHAVIOR
o--
I TI
)
m
.,-,
trial percentage preference comparisons at this concentration: 18%, 46%, and 57% for the capsaicin-treated, vehicle-treated and untreated groups, respectively. The overall curve shapes and group relations for percentage preference mirrored the standardized intake results. It may be seen in Fig. 3 that increasing sucrose concentration elicited incremental intake for all groups, F(2, 36) = 6.52, p < 0.01. No differences were observed between the two female control groups, F(1, 18) = 2.63, p > 0.05. However, the response to increasing sucrose concentrations was significantly muted in the capsaicin-treated group, F(1, 18) = 7.61; p < 0.05. In contrast, when within-trial percentage preference was considered, these groups were fairly similar: both the capsaicin-treated and the combined control groups expressed slight (i.e., 77% and 71%, respectively) sucrose preference at the lowest concentration (10 raM), each increasing preferences to a maximum (93% and 98%, respectively) at 300 raM. This suggests that the capsaicin-treated group tasted and preferred sucrose solutions, but was not as prone to sweet-induced polydipsia as the control groups. Likewise, neonatal capsaicin treatment significantly diminished consumption of sodium chloride solutions, /7(1, 18) = 8.57, p < 0.01, especially at the preferred concentrations between 30 and 300 m M (see Fig. 4). The concentration and interaction effects were also significant, F(1, 90) = 39.16, p < 0.01; F(5, 90) = 8.60, p < 0.01, respectively, while the differences between the two control groups was not, F(I, 18) = 0.29, p > 0.05. Twobottle, 48-h percentage preference comparisons yielded similar results, although the group differences were not nearly as dramatic. Capsaicin-treated and control groups showed increasing preferences (from 77% and 75% at 3 raM, up to 90% and 96% at 100 mM, respectively) peaking at 100 m M with the control group always slightly preferring more except at the lowest (3 raM) concentration. Both groups decreased preference at 300 m M (capsaicin-treated group = 24%; control groups = 56%). Thus, although the salt preference function seemed maintained for both measures, the capsaicin-treated group expressed less
CONTROL (F+M) o--- CAPSAICIN (F) ' VEHICLE (F+M)
[
25-
1039
n
=u=n
20-
"o154)
._ "o10L.
"O
eW
5-
O
.
.
.
.
.
.
.
.001
|
.
.
.
.
.
.
.
.01
.
!
.1
Capsaicin Concentration
(raM)
FIG. 1. Standardized intake of four concentrations ofeapsaicin solutions by eapsaicin-treated (n = 10), vehicle-treated (n = 10), and untreatedcontrol (n = 10) groups. The dotted line represents the expected water intake. Vertical lines are standard errors; F--females; M--males.
All groups responded to increasing quinine concentrations by progressively decreasing intake of these solutions (Fig. 2) and this effect was significant, F(4, 108) = 11.09, p < 0.05. No group differences nor interactions were evident, F(2, 27) = 3.06; F(1, 108) = 1.45, p > 0.05, respectively. If anything, the neonatally capsaicin-treated group began to avoid quinine at a lower concentration (10 #M), and this was likewise reflected in within-
I
.~
30
•""
4) t~ e" m
"0
4) N
25
CONTROL(F+M) CAPSAICIN(F) VEHICLE (F+M) H20
i
20
15"
| m
"0 I,,, t~ "0
u)
10"
l
5 . . . . . . . .
!
. . . . . . . .
!
. . . . . . . .
!
.1 1 I0 IO0 Quinine Concentration
......
!
1000
(.M)
FIG. 2. Standardized intake of five concentration of quinine solutions by eapsaicin-treated (n = 10), vehicle-treated (n = 10) and untreated-control (n = 10) groups. The dotted line represents the expected water intake. Vertical fines are standard errors; F--females; M~males.
1040
GANCHROW, SELTZER AND BIICHA('iqO
CAPSAICIN (F) 1 VEHICLE+CONTROL (F)/ H2
" +
,I,, ..........
175 G)
~150 ~ 125 .o100 |
N 75 m
50 "o
C m
25
................................. .
1
.
.
.
.
.
.
.
!
.
.
.
.
.
10
.
.
.
i
100
.
.
.
.
.
.
.
.
|
1000
Sucrose Concentration (mM)
FIG. 3. Standardized intake of three concentrations of sucrose solutions by capsaicin-treated(n = 10) and vehicle-treatedand untreated-control (n = 10) females(F). The dotted line representsthe expected water intake. Vertical lines are standard errors.
polydipsia relative to their usual drinking behavior than did the combined control groups. Following the 10 days of capsaicin drinking there was no decrease in sensitivity to 50 e M QHCI. Standardized intake averages of the quinine solution before the series of capsaicin solutions were 8 --- 3, 19 + 5, and 6 + 3, while after the series the values obtained were 8 + 2, 9 + 3, and 5 -+ 3 for capsaicintreated, vehicle, and control groups, respectively. No correlation was found between amount ofcapsaicin consumed and quinine drinking thereafter.
creased behavioral sensitivity to quinine at 6-7 weeks postinjection that was reversed by I 1- 14 weeks. Another recent study (47) reported significantly diminished chorda tympani integrated neural responses to sucrose, quinine, sodium chloride, and citric acid in neonatally capsaicin-treated rats at 8 weeks that reverted back to normal after 16 weeks. Perhaps neonatal capsaicin treatment initially depletes some taste bud neuropeptide [e.g., calcitonin gene-related peptide (CGRP)-containing fibers (34) or substance 13(47)] and ensuing progressive reinnervation during development affords recovery of more normal gustatory function with maturity. The well-established presence of mainly trigeminal fibers supplying intra- and perigemmal CGRP and substance P (10,26,35,36,39,40,42,43,49,53,54) implicates them as potential contributors to the transduction process or bud maintenance function. In mature animals, lingually injected capsaicin does eliminate substance P in taste buds, creating a decrease in intracellular serotonin (49), and systemically injected capsaicin diminishes both substance P and CGRP immunoreactive fibers in taste papillae (26). However, no measurable change in substance P content within adult taste buds has been reported following neonatal capsaicin treatment (20,40), although perigemmal depletion was substantial. Other neurosubstances in taste buds have not been examined in this context. Reversible damage could explain why neonatal capsaicin treatment failed to affect quinine aversion, but could not account for the apparent failure to express polydipsia to normally preferred substances. The general shape of the preference-aversion function for any given taste stimulus was similar across groups and did not shift along the stimulus intensity axis as a result of capsaicin treatment. However, polydipsia to highly preferred concentrations of sodium chloride and sucrose concentrations (around 100 m M for both substances) was suppressed in neonatally capsaicin-injected rats as compared to control animals, even though within-trial percentage preferences were similar across groups. It would be difficult to ascribe this depression in palatability to failure of salt or sweet information transfer due
I
DISCUSSION
Neonatal capsaicin treatment markedly diminished extraoral sensitivity to noxious heat and intraoral capsaicin stimulation suggesting small-diameter primary afferents mediating pain or pungent sensitivity sustained substantial damage, thus confirming previous studies [e.g., (19,25,33,41)]. Nonetheless, the present results show that neonatal capsaicin treatment does not affect quinine solution intake across a broad range of concentrations (see Fig. 2). If a large percentage of small diameter (C- and A~-) fibers were eliminated by this procedure, the present findings would suggest that small diameters are not specific to bitter. On the other hand, if the small diameter fibers were nonspecific, their loss might be expected to result in a decrease of neural mass reflecting a decrease in total amount of neural activity for all stimuli tasted [see e.g., (46)]. This should be expressed as increased detection and preference thresholds across all stimuli and concentrations, and yet this also was not corroborated by our results (Figs. 2-4). It is possible that unmyelinated gustatory axons are refractory to neonatal capsaicin treatment [see e.g., (18)] or that there is some recovery during development. The rats in the present study were about 10 months old at the time of quinine testing. A recent preliminary report (34) argues that the neurotoxic effect of neonatal capsaicin in the gustatory system occurs early in development, but is reversible. These authors reported a de-
100
..... 7
CAPSAICIN(F) 1 CONTROL+VEHICLE(F)
...........
H20
80
_c 60 "0
._. 40 L.
"o 20 C
~
0
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1 10 100 NaCI C o n c e n t r a t i o n
.
.
.
.
.
.
.
1000 (mM)
FIG. 4. Standardized intake of six concentrations of sodium chloride solutionsby capsaicin-treated(n = 10) and vehicle-treatedand untreatedcontrol (n = 10) females (F). The dotted line represents the expected water intake. Vertical lines are standard errors.
CAPSAICIN TREATMENT AND TASTE BEHAVIOR
to peripheral sensory afferent loss, because only the most preferred concentrations of NaC1 and sucrose were affected. Furthermore, several lines of evidence suggest that unmyelinated fibers are probably not specific for the peripheral neural representation of salt. For instance, fiber thickness and conduction velocity are related and the relatively faster reaction times [e.g., (3)] and conduction velocities [e.g., (23,24)] are elicited by sodium chloride. If there was a stimulus-fiber size linkage, these results would suggest that sodium chloride information was carried by larger diameter fibers which should not have been damaged by neonatal capsaicin treatment. Likewise, a related trigeminal-mediated sensation probably played no role in suppressing polydipsia. While higher concentrations of NaCl are known to have an irritant component (15), these are mainly expressed above 300 mM, beyond the preferred ranges under discussion. Indirectly, however, small diameter fiber loss early in development may have contributed to our results via interactions with neurons arriving to medullary regions involved with preference behaviors. Likewise, temporary elimination of critical intragemmal neurosubstances (see above) neonatally could have profound effects on the developing nucleus of the solitary tract (NST). Evidence exists that net afference in peripheral gustatory pathways during a critical period in development may mediate terminal field development at the first synaptic locus. It has been demonstrated (29) that rat fungiform taste bud damage on postnatal day 2 alters the normal development of chorda tympani/ greater superficial petrosal nerve terminal fields in the gustatory zone of the NST. Receptor damage later in development doesn't produce this reorganization which persists into adulthood long after receptor recovery. Interestingly, the correlated behavioral effect evinced in adulthood by these rats was a significantly diminished preference for concentrations of NaC1 and sucrose up to 100 mM, while percentage preference to concentrations of quinine and citric acid were not affected. Capsaicin treatment in the present experiment occurred precisely during this critical period of NST development which coincidentally marks the beginning of a 3-week period of taste bud maturation, myelinization of peripheral gustatory axons, and the development of mature neurophysiological responses to gustatory stimuli [e.g., (8,11,17,22,38,52)]. The potential effect of neonatal capsaicin treatment in the gustatory system could have been twofold: 1. There may have been some vehicle-induced taste receptor damage around postnatal day 2, because the vehicle is a detergent and has been implicated in taste bud degeneration (1);
1041 2. The hypothesized damage to gustatory neurons might have produced a temporary delay in functional receptor cell innervation due either to outright fiber degeneration or depletion of some critical neurosubstance. These, in turn, may have caused more permanent reorganization in central gustatory relays as demonstrated for NST (29). Alternatively, failure of capsaicin treatment to induce polydipsia to preferred taste solutions may have been directly related to altered Na ÷ and/or fluid regulatory functions. If fluids (and/ or Na +) from initially avid solution intake were retained in capsaicin-treated animals, feedback may have inhibited further inflated fluid intake. Indeed, neonatally capsaicin-treated rats have been shown to have lower Na ÷ and urine volume excretions than control animals following furosemide treatment (37) and this may, in part, be related to impairment of primary afferent fibers mediating full bladder sensation (21). If this mechanism were relevant, no group differences in polydipsia during the initial minutes of drinking should be expressed, because these mechanisms would not yet be operational. Behavioral studies in humans have also revealed diminished taste perceptions following oral mucosal rinses with capsaicin and related compounds (5,30,31), although the effect may be procedurally dependent (5). Neither salt nor bitter perceptions were uniquely affected in these studies. In conclusion, neonatal capsaicin treatment did not alter the normal aversion to drinking increasing quinine concentrations. The treatment seemed effective because these same rats exhibited reduced sensitivity to capsaicin solutions in the 2-bottle choice paradigm, as well as diminished nocifensive responses to laser stimulation. This implies that the smallest diameter fibers were markedly affected, and that these fibers play a rather small role in conveying quinine information. It is possible that the apparent depression in consumption of preferred substances observed in the present study results from capsaicin-induced changes in putative peptide neurotransmitter systems, altered information transfer at central relay nuclei during a critical stage in taste bud development, and/or changes in systemic fluid/Na ÷ regulatory mechanisms. ACKNOWLEDGEMENTS The authors would like to express their appreciation to Steve Brown, Vered Dor, Raziel Haimi-Cohen, Dan Oppenheimer, Heidi Siegel, Jan Stoepel, and Sylvia Kachalsky for their assistance in various aspects of this study. Funds supporting this research came from intramural sources of the Hebrew University-Hadassah Faculty of Dental Medicine as well as the Charles E. Smith Foundation. Results of this study are based on a D.M.D. Thesis (N.B.) submitted as partial fulfillment of requirements of the Hebrew University-Hadassah Faculty of Dental Medicine.
REFERENCES 1. Bardach, J. E.; Fujiya, M.; Holl, A. Detergents: Effects on the chemical senses of the fish Ictalurus natalis. Science 148:1605-1607; 1965. 2. Boudreau, J. C.; Alev, N. Classification of chemoreceptive tongue units of the cat geniculate ganglion. Brain Res. 54:157-175; 1973. 3. Bujas,Z.; Pirron, H. Le temps de rraction aux excitations gustatives d'intensit6 differrnte. C. R. Soc. Biol. (Paris) 119:1360-1362; 1935. 4. Cohen, E.; Lieblieh, I.; Ganchrow, J. R. Saccharin preferences in prepubertal female rats: Relation to self-stimulation. Behav. Neural Biol. 36:88-93; 1982. 5. Cowart, B. J. Oral chemical irritation: Does it reduce perceived taste intensity? Chem. Senses 12:467--479; 1987. 6. Diamant, H.; Zotterman, Y. A comparative study on the neural and psychophysical response to taste stimuli. In: Pfaffmann, C., ed. Olfaction and taste, vol. 2. New York: Rockefeller University Press; 1969:428-435.
7. Edwards, A. L. Statistical methods for the behavioral sciences. New York: Rinehart & Co., Inc.; 1956:315-365. 8. Farbman, A. I. Electron microscopic study of the developing taste bud in the rat fungiform papilla. Dev. Biol. l 1:I 10-135; 1965. 9. Farbman, A. I.; Hellekant, G. Quantitative analyses of the fiber population in rat chorda tympani nerves and fungiform papillae. Am. J. Anat. 153:509-522; 1978. 10. Finger, T. E. Peptide immunohistochemistry demonstrates multiple classes of perigemmal nerve fibers in the circumvallate papilla of the rat. Chem. Senses 11:135-144; 1986. 11. Ferrell, F.; Tsuetaki, T.; Chole, R. A. Myelination in the chorda tympani of postnatal rat: A quantitative electron microscope study. Acta Anat. 123:224-229; 1985. 12. Frank, M. E.; Contreras, R. J.; Hettinger, T. P. Nerve fibers sensitive to ionic taste stimuli in the chorda tympani of the rat. J. Neurophysiol. 50:941-960; 1983.
1042
13. Ganchrow, J. R. Consummatory responses to taste stimuli in the hedgehog (Erinaceus europaeus). Physiol. Behav. 18:447-453; 1977. 14. Ganchrow, J. R.; Lieblich, I.; Cohen, E. Consummatory responses to taste stimuli in rats selected for high and low rates of self-stimulation. Physiol. Behav. 27:971-976:1981. 15. Green, B. G. NaCI as an oral irritant. Chem. Senses 13:692; 1988. 16. Hanamori, T.; Ishiko, N. Conduction velocity of the lXth nerve fibers innervating taste organs in the rostral and caudal tongue region in bullfrog. Chem. Senses 6:175-187; 1981. 17. Hill, D. L.; Mistretta, C. M.; Bradley, R. M. Developmental changes in taste response characteristics of rat single chorda tympani fibers. J. Neurosci. 2:782-790; 1982. 18. Hiura, A.: Ishizuka, H.; Sakamoto, Y. Electron microscopic study of the effect of capsaicin on the mouse chorda tympani nerves. Arch. Oral Biol. 35:913-916; 1990. 19. Holje, L.; Hilderbrand, C.; Fried, K. Proportion of unmyelinated axons in the rat inferior alveolar nerve and mandibular molar pulps after neonatal administration of capsaicin. Brain Res. 266:133-136; 1983. 20. Holzer, P.; Bucsics, A.; Lembeck, F. Distribution of capsaicin-sensitive nerve fibres containing immunoreactive substance P in cutaneous and visceral tissues of the rat. Neurosci. Lett. 31:253-257; 1982. 21. Holzer-Petsche, U.; Lembeck, F. Systemic capsaicin treatment impairs the micturition reflex in the rat. Br. J. Pharmacol. 83:935-941; 1984. 22. Hosley, M. A.; Oakley, B. Postnatal development of the vallate papilla and taste buds in rats. Anat. Rec. 218:216-222; 1987. 23. lggo, A.; Leek, B. F. The afferent innervation of the tongue of the sheep. In: Hayashi, T., ed. Olfaction and taste, vol. 2. New York: Pergamon Press; 1967:493-508. 24. Iriuchijima, J.; Zotterman, Y. Conduction rates of afferent fibers to the anterior tongue of the dog. Acta Physiol. Scand. 51:283-289; 1961. 25. Janc~o, G.; Kiraly, E.; Janc~o-G'abor, A. Pharmacologically induced selective degeneration of chemosensitive primary sensory neurones. Nature 270:741-743; 1977. 26. Kinnman, E.; Aldskogius, H. The role of substance P and calcitonin gene-related peptide containing nerve fibers in maintaining fungiform taste buds in the rat after a chronic chorda tympani nerve injury. Exp. Neurol. 113:85-91; 1991. 27. Kitchell, R. L. Comparative anatomical and physiological studies of gustatory mechanisms. In: Zotterman, Y., ed. Olfaction and taste. vol 1. New York: The MacMillan Co.; 1963:235-291. 28. Ki'ecek, J. Sex differences in taste preference for a salt solution in the rat. Physiol. Behav. 8:183-188; 1972. 29. Lasiter, P. S.; Kachele, D. L. Effects of early postnatal receptor damage on development of gustatory recipient zones within the nucleus of the solitary tract. Dev. Brain Res. 55:57-71 ; 1990. 30. Lawless, H.; Stevens, D. A. Effects of oral chemical irritation on taste. Physiol. Behav. 32:995-998, 1984. 31. Lawless, H.; Rozin, P.; Shenker, J. Effects of oral capsaicin on gustatory, olfactory and irritant sensations and flavor identification in humans who regularly or rarely consume chili pepper. Chem. Senses 10:579-589; 1985. 32. Lawson, J. W.; Gomez, M. M.; Miller, 1. J. Taste sensitivity and primary axon morphology. Anat. Rec. 193:601; 1979. 33. Lawson, S. N.; Nickels, S. M. The use of morphometric techniques to analyse the effect of neonatal capsaicin treatment on rat dorsal root ganglia and dorsal roots. J. Physiol. (Loud.). 303:12P; 1980. 34. Lindstrand, K.; Enslen, M. The effect of capsaicin on taste. Chem. Senses 16:547; 1991. 35. Lundberg, J. M.; Hokfelt, T.; Angard, A.; Pernow, B.; Emson, P. lmmunohistochemical evidence for substance P immunoreactive nerve fibers in the taste buds of the cat. Acta Physiol. Scand. 107: 389-391: 1979.
GANCHROW,
SELTZER AND BII(THACH()
36. Luts, A.; Montavon, P.; Lindstrand, K., Sundler, [-. Peptide-conraining nerve fibers in the circumvallate papillae. Reg. Pept. 27:209226: 1990. 37. Massi, M.; Polidori, C.; Perfumi, M.; Ciccocioppo, R.; DeCaro, G.; Bacciarelli, C.; Manzini, S. Effect of capsalcin neonatal treatment on the salt intake of the adult rat. Pharmacol. Biochem. Behav. 40: 163-168, 1991. 38. Mistretta, C. M. Topographical and histological study of the developing rat tongue, palate and taste buds. In: Bosma, J. F., ed. Oral sensation and perception, vol. 3: The mouth of the infant. New York: Thomas; 1972:163-167. 39. Montavon, Ph.; Lindstrand, K.; Luts, A.; Sundler, F. Peptide-containing nerve fibers in the fungiform papillae of pigs and rats. Reg. Pept. 32:141-150; 1991. 40. Nagy, J. I.; Goedert, M.; Hunt, S. P.; Bond, A. The nature of the substance P-containing nerve fibres in taste papillae of the rat tongue. Neuroscience 7:3137-3151; 1982. 41. Nagy, J. 1.; Iverson, L. L.; Goedert, M.; Chapman, D.; Hunt, S. P. Dose-dependent effects of capsaicin on primary sensory neurons in the neonatal rat. J. Neurosci. 3:399-406; 1983. 42. Nishimoto, T.; Akai, M.; Inagaki, S.; Shiosaka, S.; Shimizu, Y.; Yamamoto, K.; Senba, E.; Sakanaka, M.; Takatsuki, K.; Hara, Y.; Takagi, H.; Matsuzaki, T.; Kawai, Y.; Tohyama, M. On the distribution and origins of substance P in the papillae of the tongue: An experimental and immunohistochemical study. J. Comp. Neurol. 207:85-92; 1982. 43. Nishimoto, T.; Ichikawa, H.; Wakisaka, S.; Matsuo, S.; Yamamoto, K.; Nakata, T.; Akai, M. Immunohistochemical observation on substance P in regenerating taste buds of the rat. Anat. Rec. 212: 430-436; 1985. 44. Nowlis. G. H.; Frank, M. Qualities in hamster taste: Behavioral and neural evidence. In: LeMagnen, J.; Macleod, P., eds. Olfaction and taste, vol. 4. London: Information Retrieval, Inc.; 1977:241248. 45. Scadding, J. W. The permanent anatomical effects of neonatal capsaicin on somatosensory nerves. J. Anat. 131:473-484; 1980. 46. Schiffman, S.; Orlandi, M.; Erickson, R. P. Changes in taste and smell with age: Biological aspects. In: Ordy, J. M.; Brizzee, K., eds. Sensory systems and communication in the elderly, vol. 10. Aging. New York: Raven Press; 1979:247-267. 47. Serova, O. N. Changes in activity of the taste receptor apparatus and preference for sodium chloride solution in newborn rats treated with capsaicin. Bull. Exp. Biol. Med. 109:269-271; 1990. 48. Silver, W. L.; Mason, J. R.; Marshall, D. A.; Maruniak, J. A. Rat trigeminal, olfactory and taste responses after capsaicin densensitization. Brain Res. 333:45-54; 1985. 49. Solov'eva, N. A.; Esakov, A. I. Pharmaco-histochemical study of monoamine-containing taste bud cells in capsaicin-induced substance P deficiency. Bull. Exp. Biol. Med. (English Translation). 102:1601 1603; 1986. 50. Valenstein, E. S.; Cox, V. C.; Kakolewski, J. W. Further studies of sex differences in taste preferences with sweet solutions. Psychol. Rep. 20:1231-1234; 1967. 51. Wirtshafter, D.; Asin, K. E. A comment on the practice of expressing food and water intake as proportion of body weight. Physiol. Behav. 28:183-185; 1982. 52. Yamada, T. Chorda tympani responses to gustatory stimuli in developing rats. Jpn. J. Physiol. 30:631-654; 1980. 53. Yamasaki, H.; Kubota, Y.; Takagi, H.; Tohyama, M. Immunoelectronmicroscopic study on the fine structure of substance P-containing fibers in the taste buds of the rat. J. Comp. Neurol. 227:380-392; 1984. 54. Yamasaki, H.; Kubota, Y.; Tohyama, M. Ontogeny of substance-P containing fibers in the taste buds and surrounding epithelium. I. Light microscopic analysis. Dev. Brain. Res. 18: 301-305; 1985.
0031-9384/92 $5.00 + .00 Copyright© 1992PergamonPressLtd.
Printed in the USA.
The Effect of Neonatal Capsaicin Treatment on Gustatory Behavior in the Albino Rat JUDITH
R. G A N C H R O W , .1 Z E ' E V S E L T Z E R * A N D N I T Z A N BITCHACHO~"
*The Hebrew University-Itadassah Faculty of Dental Medicine Founded by the Alpha Omega Fraternity, Jerusalem, Israel and y'The Goldshlager Faculty of Dental Medicine, Tel Aviv University, Tel Aviv, Israel R e c e i v e d 12 M a r c h 1992 GANCHROW, J. R., Z. SELTZER AND N. BITCHACHO. The effect of neonatal capsaicin treatment on gustatorybehaviorin the albino rat. PHYSIOL BEHAV 52(6) 1037-1042, 1992.--Small-diameter fibers present in gustatory peripheral nerves have historically been suspected of relaying information about the bitter quality of a taste stimulus. Neonatally injected capsaicin irreversibly destroys a proportion of unmyelinated C- and some A~-fibers.Consummatory responses to increasing concentrations of quinine and other chemical solutions following neonatal capsaicin injection were compared to those of untreated and vehicleinjected control Sabra albino rats. Capsaicin-treated rats significantly increased their withdrawal thresholds to noxious, CO2 lasergenerated heat pulses verifyingtreatment effectiveness.Furthermore, neonatal capsaicin treatment diminished sensitivity to pungent capsaicin solutions in mature rats. However, there were no group differences in quinine intake, suggesting that the full array of unmyelinated fibers associated with taste buds is not essential for the transmission of bitter taste. Capsaicin-treated animals showed a significant reduction in intake of normally highly preferred sodium chloride and sucrose concentrations. These results were probably not due to loss of peripheral unmyelinated afferent fibers per se, but rather to secondary central changes. Capsaicin
Taste
Preference
Consummatory behavior
GUSTATORY nerves are comprised of both myelinated and unmyelinated fibers (9,27,32). Some hypotheses concerning quality coding have suggested different roles for fibers of different diameters [e.g., (2,12)]. For instance, based on size, conduction velocity and/or scarcity, it has been suggested that the smallest diameter fibers convey bitter information (6,16,23,24). Furthermore, the population of afferent (unmyelinated) C-fibers in the glossopharyngeal nerve is markedly larger than that found in the chorda tympani nerve (32), possibly correlating with the larger proportion of glossopharyngeal fibers responding to quinine [e.g., (44)]. Capsaicin (trans-8-methyl-N-vanyllyl-6-nonanamide), the pungent substance in chili peppers, is known to produce rapid, irreversible destruction of many primary sensory C- and some A~-fibers when administered neonatally at a concentration of 50 mg/kg [e.g., (19,25,33,41,45)]. If the fine afferent fibers of the gustatory nerves were specifically labelled to carry qualitatively bitter information, one would expect neonatal capsaicin treatment to severely impair preference behavior, producing heightened thresholds and overall diminished responsivity to an increasing quinine concentration series. Some support for this hypothesis has been obtained following repeated capsaicin injections in adult rats (48), who subsequently showed decreased sensitivity only to higher concentrations of quinine in preference tests that included sodium chloride and citric acid as well. The purpose of this study was to examine the behavioral responses of adult rats to several concentrations of bitter, sweet,
Quinine
Sucrose
NaC1
Development
salty, and pungent stimuli, following a single neonatal capsaicin treatment. METHOD
Subjects Consummatory behavior was examined in 30 Sabra strain albino rats, divided into three groups. Members of each group were drawn from eight litters born within a week of each other. On postnatal day 2, 49 pups of either sex were injected subcutaneously with a 50 mg/kg dose of capsaicin dissolved in the vehicle [10% Tween 80, 10% ethyl alcohol in saline (24)] totalling 35 #l volume. In order to prevent leakage, the syringe needle was inserted at the nape of the neck and advanced subdermally such that injections were made at the lumbar region. At the same time, a comparable group of 14 littermate pups received the vehicle alone, and 12 pups served as untreated controls. All rats were weaned at 21 days and reared under similar group conditions and taste testing began on postnatal day 290. Following a lower survival rate among males treated neonatally with capsaicin, the remaining animals were divided into groups as follows: the capsaicin-treated group was comprised often females (average weight 229 g) and initially three surviving males (average weight 490 g) who died during the early phases of the experiment, and their data are, therefore, not included. The two control groups (vehicle and untreated) each included five females (average weights 227 and 232 g, respectively) and five males
Requests for reprints should be addressed to Dr. J. R. Ganchrow, Department of Oral Biology, The Hebrew University-Hadassah, Faculty of Dental Medicine, P.O.B. 1172, Jerusalem 91010, Israel. 1037
1038 (average weights 550 and 429 g, respectively). One female in the experimental group died during the last week of testing. The females receiving capsaicin gained an average of 64 g during the 62 days of testing, while the females of the vehicle group and the normal group gained an average of 64 and 56 g, respectively. The males of the latter two groups gained an average of 108 g and 82 g, respectively.
Procedures The experiment was carried out in a room under a 13 h-light (commencing at 0700 h), 11-h dark cycle. Room temperature was maintained at 27 + 1°C. Laboratory rat chow (Ambar Ltd. 931) was available ad lib throughout the experiment. Nociceptive reflex threshold test. Seven days before the start of the experiment, each animal was tested for withdrawal response thresholds to ultra short noxious heat pulses radiated from a CO2 laser (5 watts; 30-120 ms; 40-150 mcal) applied to the ear, nose, forepaw, hindpaw, and tail. The stimulated target (1.5 mm in diameter) was illuminated by a visible spot of red light from the He/Ne-aiming laser. Heat pulse intensity was gradually increased until the unrestrained animal responded by withdrawal which was recorded via pressure-transduced oscillographic traces and confirmed visually. Drinking preference test. A standard 2-bottle, 24-h testing procedure was utilized to monitor taste-guided fluid consumption. During preference testing, each animal was housed in a 24 × 35 × 12 cm wire mesh cage, suspended on a rack. Members of each group were randomly distributed into the bank of cages and their identity concealed such that data were recorded under blind conditions. Two 100 ml graduated glass cylinders fitted with rubber stoppers and L-shaped, drinking spouts were affixed to the front of each cage, about 4 cm from each lateral edge (separation distance = 16 cm), and 4 cm above the floor. A food tray occupied a position midway between the two drinking tubes. The tapering tip of each stainless steel drinking spout (inside tip diameter about 1.5 mm) inserted to about 2 cm inside the cage for fluid delivery. Each stimulus concentration was presented for choice against bidistilled water for a total of 48 h, 24 h on each side to balance out side preferences. Readings of the fluid volume contained in each bottle were taken to the nearest ml between 1115 and 1130 h each morning, and fluids were then either replenished or replaced, and their volumes again recorded. Solutions were prepared from reagent grade chemicals, and bidistilled water was the solvent. The test stimuli included sucrose (Suc) at 10, 30, and 300 mM; quinine hydrochloride (QHC1) at 1, 5, 10, 50, and 500 uM,' sodium chloride (NaCI) at 3, 10, 30, 100, 300, and 500 mM; and eapsaiein (Cap) dissolved in 100 ul ethanol (ETOH) per liter of water at 0.0016, 0.0023, 0.0033, and 0.016 mM. Since the capsaicin was by necessity dissolved in ethanol, 100 ul ETOH was likewise included in each liter of water presented for choice against the capsaicin solutions. This miniscule amount of ETOH in the distilled water was not detectable to the experimenters. Stimuli were presented in ascending concentrations in the above order with the addition of 50 # M QHC1 before and immediately following the capsaicin run, to observe whether drinking capsaicin in the days preceding quinine might diminish sensitivity to the latter. Three days of water available in both bottles interceded between each change in chemical category with the exception of the last quinine exposure, which occurred on the day immediately following the highest capsaicin concentration.
Data Anal.vsis Intake measures were standardized relative to the amount of fluid an individual rat would be likely to consume in 24 h from
GANCHROW, SELTZER AND BII'CttACHO one bottle, if both bottles contained water. Thus, mean 24-h solution intake was compared to the averaged daily one-bottle water intake for the 3 days immediately preceding and the 3 days immediately following any stimulus concentration series for each animal. Single bottle water intake is defined as half the total daily two-bottle water intake. Therefore, standard intake = [mean (2 day) solution intake/mean (6 day) single bottle water intake] × 20. This sets each rat's single bottle water intake at 20 and expresses all other intakes relative to this value. (The value 20 was arbitrarily chosen since the average 24-h water consumption in normal Sabra rats is around 40 ml, or theoretically 20 ml/bottle.) Thus, values greater than 20 would suggest a tendency toward preference, and less than 20, aversion. This transformation was utilized in an attempt to decrease the variability contributed by different fluid consumptions between individuals or between groups. It was deemed to be a more sensitive measure of taste reactivity than the usual within-trial two-bottle, 24-h percentage measure (percentage preference = [solution intake/ total intake] X 100%), where consumption from each bottle may be differentially affected both by taste and by physiological reactions, such as water regulation [see e.g., (13)] influenced by a variety of sources. Furthermore, this method avoids the problems of standardizing by animal weight [see (51)]. A new multiplication factor was obtained for each chemical category tested, to control for possible shifts in fluid consumption over the 2 months of testing, due to possible changes in seasonal climatic condition, animal aging, or other unknown factors. All group data are presented as means __ SEM. The rejection level for analyses of variance two-factor, mixed design, (7) and other statistical procedures was p < 0.05. Males and females were grouped together for statistical analysis except when examining responses to sucrose and NaC1, substances known to elicit sex differences in rat preference behavior [e.g., (4,14,28,50)]. RESULTS Capsaicin treatment profoundly decreased sensitivity to painful stimuli in the various extraoral body sites examined. The average withdrawal threshold of capsalcin-treated rats was 40°70 higher than that of the combined control groups, and this difference was significant both for the grouped data and for between group comparisons (e.g., for individual noxious stimulus sites the threshold ranges were t = 2.65-7.78, p -< 0.0005-0.01 for the capsalcin versus control group comparisons, and t = 2.21-5.07, p -< 0.0005-0.025 for the capsaicin versus vehicle group comparisons, one-tailed t-test). Intraoral sensitivity to irritants was also affected: neonatal capsaicin treatment diminished aversion to drinking capsaicin solutions in adult female rats. It may be seen in Fig. 1 that capsaicin-treated animals continued drinking capsaicin solutions up to 0.0033 m M in amounts similar to normal water drinking while the two control groups decreased their intake by about half in these concentration ranges. The group differences were significant, F(2, 27) = 5.45, p < 0.01, as were the concentration, F(3, 81) = 8.54, p < 0.01, and the interaction, F(6, 81) = 3.85, p < 0.0 l, effects. When males of the two control groups were compared to females of the two control groups, no significant differences were found, F(1, 18) = 0.52, p > 0.05, indicating that gender is not relevant to capsaicin drinking. These relationships and curve shapes were maintained when within-trial percentage preference was calculated. For example, at 0.0033 raM, percent preference values for capsaicin solutions were 52070, 32°70, and 1207ofor the capsaicin-treated, vehicle-treated, and untreated controls, respectively. In this kind of analysis, 50070 represents indifference and decreasing values signify increasing aversion. Thus, even at 0.0033 rnM the neonatally capsaicintreated group did not refrain from drinking capsaicin solutions.
CAPSAICIN TREATMENT AND TASTE BEHAVIOR
o--
I TI
)
m
.,-,
trial percentage preference comparisons at this concentration: 18%, 46%, and 57% for the capsaicin-treated, vehicle-treated and untreated groups, respectively. The overall curve shapes and group relations for percentage preference mirrored the standardized intake results. It may be seen in Fig. 3 that increasing sucrose concentration elicited incremental intake for all groups, F(2, 36) = 6.52, p < 0.01. No differences were observed between the two female control groups, F(1, 18) = 2.63, p > 0.05. However, the response to increasing sucrose concentrations was significantly muted in the capsaicin-treated group, F(1, 18) = 7.61; p < 0.05. In contrast, when within-trial percentage preference was considered, these groups were fairly similar: both the capsaicin-treated and the combined control groups expressed slight (i.e., 77% and 71%, respectively) sucrose preference at the lowest concentration (10 raM), each increasing preferences to a maximum (93% and 98%, respectively) at 300 raM. This suggests that the capsaicin-treated group tasted and preferred sucrose solutions, but was not as prone to sweet-induced polydipsia as the control groups. Likewise, neonatal capsaicin treatment significantly diminished consumption of sodium chloride solutions, /7(1, 18) = 8.57, p < 0.01, especially at the preferred concentrations between 30 and 300 m M (see Fig. 4). The concentration and interaction effects were also significant, F(1, 90) = 39.16, p < 0.01; F(5, 90) = 8.60, p < 0.01, respectively, while the differences between the two control groups was not, F(I, 18) = 0.29, p > 0.05. Twobottle, 48-h percentage preference comparisons yielded similar results, although the group differences were not nearly as dramatic. Capsaicin-treated and control groups showed increasing preferences (from 77% and 75% at 3 raM, up to 90% and 96% at 100 mM, respectively) peaking at 100 m M with the control group always slightly preferring more except at the lowest (3 raM) concentration. Both groups decreased preference at 300 m M (capsaicin-treated group = 24%; control groups = 56%). Thus, although the salt preference function seemed maintained for both measures, the capsaicin-treated group expressed less
CONTROL (F+M) o--- CAPSAICIN (F) ' VEHICLE (F+M)
[
25-
1039
n
=u=n
20-
"o154)
._ "o10L.
"O
eW
5-
O
.
.
.
.
.
.
.
.001
|
.
.
.
.
.
.
.
.01
.
!
.1
Capsaicin Concentration
(raM)
FIG. 1. Standardized intake of four concentrations ofeapsaicin solutions by eapsaicin-treated (n = 10), vehicle-treated (n = 10), and untreatedcontrol (n = 10) groups. The dotted line represents the expected water intake. Vertical lines are standard errors; F--females; M--males.
All groups responded to increasing quinine concentrations by progressively decreasing intake of these solutions (Fig. 2) and this effect was significant, F(4, 108) = 11.09, p < 0.05. No group differences nor interactions were evident, F(2, 27) = 3.06; F(1, 108) = 1.45, p > 0.05, respectively. If anything, the neonatally capsaicin-treated group began to avoid quinine at a lower concentration (10 #M), and this was likewise reflected in within-
I
.~
30
•""
4) t~ e" m
"0
4) N
25
CONTROL(F+M) CAPSAICIN(F) VEHICLE (F+M) H20
i
20
15"
| m
"0 I,,, t~ "0
u)
10"
l
5 . . . . . . . .
!
. . . . . . . .
!
. . . . . . . .
!
.1 1 I0 IO0 Quinine Concentration
......
!
1000
(.M)
FIG. 2. Standardized intake of five concentration of quinine solutions by eapsaicin-treated (n = 10), vehicle-treated (n = 10) and untreated-control (n = 10) groups. The dotted line represents the expected water intake. Vertical fines are standard errors; F--females; M~males.
1040
GANCHROW, SELTZER AND BIICHA('iqO
CAPSAICIN (F) 1 VEHICLE+CONTROL (F)/ H2
" +
,I,, ..........
175 G)
~150 ~ 125 .o100 |
N 75 m
50 "o
C m
25
................................. .
1
.
.
.
.
.
.
.
!
.
.
.
.
.
10
.
.
.
i
100
.
.
.
.
.
.
.
.
|
1000
Sucrose Concentration (mM)
FIG. 3. Standardized intake of three concentrations of sucrose solutions by capsaicin-treated(n = 10) and vehicle-treatedand untreated-control (n = 10) females(F). The dotted line representsthe expected water intake. Vertical lines are standard errors.
polydipsia relative to their usual drinking behavior than did the combined control groups. Following the 10 days of capsaicin drinking there was no decrease in sensitivity to 50 e M QHCI. Standardized intake averages of the quinine solution before the series of capsaicin solutions were 8 --- 3, 19 + 5, and 6 + 3, while after the series the values obtained were 8 + 2, 9 + 3, and 5 -+ 3 for capsaicintreated, vehicle, and control groups, respectively. No correlation was found between amount ofcapsaicin consumed and quinine drinking thereafter.
creased behavioral sensitivity to quinine at 6-7 weeks postinjection that was reversed by I 1- 14 weeks. Another recent study (47) reported significantly diminished chorda tympani integrated neural responses to sucrose, quinine, sodium chloride, and citric acid in neonatally capsaicin-treated rats at 8 weeks that reverted back to normal after 16 weeks. Perhaps neonatal capsaicin treatment initially depletes some taste bud neuropeptide [e.g., calcitonin gene-related peptide (CGRP)-containing fibers (34) or substance 13(47)] and ensuing progressive reinnervation during development affords recovery of more normal gustatory function with maturity. The well-established presence of mainly trigeminal fibers supplying intra- and perigemmal CGRP and substance P (10,26,35,36,39,40,42,43,49,53,54) implicates them as potential contributors to the transduction process or bud maintenance function. In mature animals, lingually injected capsaicin does eliminate substance P in taste buds, creating a decrease in intracellular serotonin (49), and systemically injected capsaicin diminishes both substance P and CGRP immunoreactive fibers in taste papillae (26). However, no measurable change in substance P content within adult taste buds has been reported following neonatal capsaicin treatment (20,40), although perigemmal depletion was substantial. Other neurosubstances in taste buds have not been examined in this context. Reversible damage could explain why neonatal capsaicin treatment failed to affect quinine aversion, but could not account for the apparent failure to express polydipsia to normally preferred substances. The general shape of the preference-aversion function for any given taste stimulus was similar across groups and did not shift along the stimulus intensity axis as a result of capsaicin treatment. However, polydipsia to highly preferred concentrations of sodium chloride and sucrose concentrations (around 100 m M for both substances) was suppressed in neonatally capsaicin-injected rats as compared to control animals, even though within-trial percentage preferences were similar across groups. It would be difficult to ascribe this depression in palatability to failure of salt or sweet information transfer due
I
DISCUSSION
Neonatal capsaicin treatment markedly diminished extraoral sensitivity to noxious heat and intraoral capsaicin stimulation suggesting small-diameter primary afferents mediating pain or pungent sensitivity sustained substantial damage, thus confirming previous studies [e.g., (19,25,33,41)]. Nonetheless, the present results show that neonatal capsaicin treatment does not affect quinine solution intake across a broad range of concentrations (see Fig. 2). If a large percentage of small diameter (C- and A~-) fibers were eliminated by this procedure, the present findings would suggest that small diameters are not specific to bitter. On the other hand, if the small diameter fibers were nonspecific, their loss might be expected to result in a decrease of neural mass reflecting a decrease in total amount of neural activity for all stimuli tasted [see e.g., (46)]. This should be expressed as increased detection and preference thresholds across all stimuli and concentrations, and yet this also was not corroborated by our results (Figs. 2-4). It is possible that unmyelinated gustatory axons are refractory to neonatal capsaicin treatment [see e.g., (18)] or that there is some recovery during development. The rats in the present study were about 10 months old at the time of quinine testing. A recent preliminary report (34) argues that the neurotoxic effect of neonatal capsaicin in the gustatory system occurs early in development, but is reversible. These authors reported a de-
100
..... 7
CAPSAICIN(F) 1 CONTROL+VEHICLE(F)
...........
H20
80
_c 60 "0
._. 40 L.
"o 20 C
~
0
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1 10 100 NaCI C o n c e n t r a t i o n
.
.
.
.
.
.
.
1000 (mM)
FIG. 4. Standardized intake of six concentrations of sodium chloride solutionsby capsaicin-treated(n = 10) and vehicle-treatedand untreatedcontrol (n = 10) females (F). The dotted line represents the expected water intake. Vertical lines are standard errors.
CAPSAICIN TREATMENT AND TASTE BEHAVIOR
to peripheral sensory afferent loss, because only the most preferred concentrations of NaC1 and sucrose were affected. Furthermore, several lines of evidence suggest that unmyelinated fibers are probably not specific for the peripheral neural representation of salt. For instance, fiber thickness and conduction velocity are related and the relatively faster reaction times [e.g., (3)] and conduction velocities [e.g., (23,24)] are elicited by sodium chloride. If there was a stimulus-fiber size linkage, these results would suggest that sodium chloride information was carried by larger diameter fibers which should not have been damaged by neonatal capsaicin treatment. Likewise, a related trigeminal-mediated sensation probably played no role in suppressing polydipsia. While higher concentrations of NaCl are known to have an irritant component (15), these are mainly expressed above 300 mM, beyond the preferred ranges under discussion. Indirectly, however, small diameter fiber loss early in development may have contributed to our results via interactions with neurons arriving to medullary regions involved with preference behaviors. Likewise, temporary elimination of critical intragemmal neurosubstances (see above) neonatally could have profound effects on the developing nucleus of the solitary tract (NST). Evidence exists that net afference in peripheral gustatory pathways during a critical period in development may mediate terminal field development at the first synaptic locus. It has been demonstrated (29) that rat fungiform taste bud damage on postnatal day 2 alters the normal development of chorda tympani/ greater superficial petrosal nerve terminal fields in the gustatory zone of the NST. Receptor damage later in development doesn't produce this reorganization which persists into adulthood long after receptor recovery. Interestingly, the correlated behavioral effect evinced in adulthood by these rats was a significantly diminished preference for concentrations of NaC1 and sucrose up to 100 mM, while percentage preference to concentrations of quinine and citric acid were not affected. Capsaicin treatment in the present experiment occurred precisely during this critical period of NST development which coincidentally marks the beginning of a 3-week period of taste bud maturation, myelinization of peripheral gustatory axons, and the development of mature neurophysiological responses to gustatory stimuli [e.g., (8,11,17,22,38,52)]. The potential effect of neonatal capsaicin treatment in the gustatory system could have been twofold: 1. There may have been some vehicle-induced taste receptor damage around postnatal day 2, because the vehicle is a detergent and has been implicated in taste bud degeneration (1);
1041 2. The hypothesized damage to gustatory neurons might have produced a temporary delay in functional receptor cell innervation due either to outright fiber degeneration or depletion of some critical neurosubstance. These, in turn, may have caused more permanent reorganization in central gustatory relays as demonstrated for NST (29). Alternatively, failure of capsaicin treatment to induce polydipsia to preferred taste solutions may have been directly related to altered Na ÷ and/or fluid regulatory functions. If fluids (and/ or Na +) from initially avid solution intake were retained in capsaicin-treated animals, feedback may have inhibited further inflated fluid intake. Indeed, neonatally capsaicin-treated rats have been shown to have lower Na ÷ and urine volume excretions than control animals following furosemide treatment (37) and this may, in part, be related to impairment of primary afferent fibers mediating full bladder sensation (21). If this mechanism were relevant, no group differences in polydipsia during the initial minutes of drinking should be expressed, because these mechanisms would not yet be operational. Behavioral studies in humans have also revealed diminished taste perceptions following oral mucosal rinses with capsaicin and related compounds (5,30,31), although the effect may be procedurally dependent (5). Neither salt nor bitter perceptions were uniquely affected in these studies. In conclusion, neonatal capsaicin treatment did not alter the normal aversion to drinking increasing quinine concentrations. The treatment seemed effective because these same rats exhibited reduced sensitivity to capsaicin solutions in the 2-bottle choice paradigm, as well as diminished nocifensive responses to laser stimulation. This implies that the smallest diameter fibers were markedly affected, and that these fibers play a rather small role in conveying quinine information. It is possible that the apparent depression in consumption of preferred substances observed in the present study results from capsaicin-induced changes in putative peptide neurotransmitter systems, altered information transfer at central relay nuclei during a critical stage in taste bud development, and/or changes in systemic fluid/Na ÷ regulatory mechanisms. ACKNOWLEDGEMENTS The authors would like to express their appreciation to Steve Brown, Vered Dor, Raziel Haimi-Cohen, Dan Oppenheimer, Heidi Siegel, Jan Stoepel, and Sylvia Kachalsky for their assistance in various aspects of this study. Funds supporting this research came from intramural sources of the Hebrew University-Hadassah Faculty of Dental Medicine as well as the Charles E. Smith Foundation. Results of this study are based on a D.M.D. Thesis (N.B.) submitted as partial fulfillment of requirements of the Hebrew University-Hadassah Faculty of Dental Medicine.
REFERENCES 1. Bardach, J. E.; Fujiya, M.; Holl, A. Detergents: Effects on the chemical senses of the fish Ictalurus natalis. Science 148:1605-1607; 1965. 2. Boudreau, J. C.; Alev, N. Classification of chemoreceptive tongue units of the cat geniculate ganglion. Brain Res. 54:157-175; 1973. 3. Bujas,Z.; Pirron, H. Le temps de rraction aux excitations gustatives d'intensit6 differrnte. C. R. Soc. Biol. (Paris) 119:1360-1362; 1935. 4. Cohen, E.; Lieblieh, I.; Ganchrow, J. R. Saccharin preferences in prepubertal female rats: Relation to self-stimulation. Behav. Neural Biol. 36:88-93; 1982. 5. Cowart, B. J. Oral chemical irritation: Does it reduce perceived taste intensity? Chem. Senses 12:467--479; 1987. 6. Diamant, H.; Zotterman, Y. A comparative study on the neural and psychophysical response to taste stimuli. In: Pfaffmann, C., ed. Olfaction and taste, vol. 2. New York: Rockefeller University Press; 1969:428-435.
7. Edwards, A. L. Statistical methods for the behavioral sciences. New York: Rinehart & Co., Inc.; 1956:315-365. 8. Farbman, A. I. Electron microscopic study of the developing taste bud in the rat fungiform papilla. Dev. Biol. l 1:I 10-135; 1965. 9. Farbman, A. I.; Hellekant, G. Quantitative analyses of the fiber population in rat chorda tympani nerves and fungiform papillae. Am. J. Anat. 153:509-522; 1978. 10. Finger, T. E. Peptide immunohistochemistry demonstrates multiple classes of perigemmal nerve fibers in the circumvallate papilla of the rat. Chem. Senses 11:135-144; 1986. 11. Ferrell, F.; Tsuetaki, T.; Chole, R. A. Myelination in the chorda tympani of postnatal rat: A quantitative electron microscope study. Acta Anat. 123:224-229; 1985. 12. Frank, M. E.; Contreras, R. J.; Hettinger, T. P. Nerve fibers sensitive to ionic taste stimuli in the chorda tympani of the rat. J. Neurophysiol. 50:941-960; 1983.
1042
13. Ganchrow, J. R. Consummatory responses to taste stimuli in the hedgehog (Erinaceus europaeus). Physiol. Behav. 18:447-453; 1977. 14. Ganchrow, J. R.; Lieblich, I.; Cohen, E. Consummatory responses to taste stimuli in rats selected for high and low rates of self-stimulation. Physiol. Behav. 27:971-976:1981. 15. Green, B. G. NaCI as an oral irritant. Chem. Senses 13:692; 1988. 16. Hanamori, T.; Ishiko, N. Conduction velocity of the lXth nerve fibers innervating taste organs in the rostral and caudal tongue region in bullfrog. Chem. Senses 6:175-187; 1981. 17. Hill, D. L.; Mistretta, C. M.; Bradley, R. M. Developmental changes in taste response characteristics of rat single chorda tympani fibers. J. Neurosci. 2:782-790; 1982. 18. Hiura, A.: Ishizuka, H.; Sakamoto, Y. Electron microscopic study of the effect of capsaicin on the mouse chorda tympani nerves. Arch. Oral Biol. 35:913-916; 1990. 19. Holje, L.; Hilderbrand, C.; Fried, K. Proportion of unmyelinated axons in the rat inferior alveolar nerve and mandibular molar pulps after neonatal administration of capsaicin. Brain Res. 266:133-136; 1983. 20. Holzer, P.; Bucsics, A.; Lembeck, F. Distribution of capsaicin-sensitive nerve fibres containing immunoreactive substance P in cutaneous and visceral tissues of the rat. Neurosci. Lett. 31:253-257; 1982. 21. Holzer-Petsche, U.; Lembeck, F. Systemic capsaicin treatment impairs the micturition reflex in the rat. Br. J. Pharmacol. 83:935-941; 1984. 22. Hosley, M. A.; Oakley, B. Postnatal development of the vallate papilla and taste buds in rats. Anat. Rec. 218:216-222; 1987. 23. lggo, A.; Leek, B. F. The afferent innervation of the tongue of the sheep. In: Hayashi, T., ed. Olfaction and taste, vol. 2. New York: Pergamon Press; 1967:493-508. 24. Iriuchijima, J.; Zotterman, Y. Conduction rates of afferent fibers to the anterior tongue of the dog. Acta Physiol. Scand. 51:283-289; 1961. 25. Janc~o, G.; Kiraly, E.; Janc~o-G'abor, A. Pharmacologically induced selective degeneration of chemosensitive primary sensory neurones. Nature 270:741-743; 1977. 26. Kinnman, E.; Aldskogius, H. The role of substance P and calcitonin gene-related peptide containing nerve fibers in maintaining fungiform taste buds in the rat after a chronic chorda tympani nerve injury. Exp. Neurol. 113:85-91; 1991. 27. Kitchell, R. L. Comparative anatomical and physiological studies of gustatory mechanisms. In: Zotterman, Y., ed. Olfaction and taste. vol 1. New York: The MacMillan Co.; 1963:235-291. 28. Ki'ecek, J. Sex differences in taste preference for a salt solution in the rat. Physiol. Behav. 8:183-188; 1972. 29. Lasiter, P. S.; Kachele, D. L. Effects of early postnatal receptor damage on development of gustatory recipient zones within the nucleus of the solitary tract. Dev. Brain Res. 55:57-71 ; 1990. 30. Lawless, H.; Stevens, D. A. Effects of oral chemical irritation on taste. Physiol. Behav. 32:995-998, 1984. 31. Lawless, H.; Rozin, P.; Shenker, J. Effects of oral capsaicin on gustatory, olfactory and irritant sensations and flavor identification in humans who regularly or rarely consume chili pepper. Chem. Senses 10:579-589; 1985. 32. Lawson, J. W.; Gomez, M. M.; Miller, 1. J. Taste sensitivity and primary axon morphology. Anat. Rec. 193:601; 1979. 33. Lawson, S. N.; Nickels, S. M. The use of morphometric techniques to analyse the effect of neonatal capsaicin treatment on rat dorsal root ganglia and dorsal roots. J. Physiol. (Loud.). 303:12P; 1980. 34. Lindstrand, K.; Enslen, M. The effect of capsaicin on taste. Chem. Senses 16:547; 1991. 35. Lundberg, J. M.; Hokfelt, T.; Angard, A.; Pernow, B.; Emson, P. lmmunohistochemical evidence for substance P immunoreactive nerve fibers in the taste buds of the cat. Acta Physiol. Scand. 107: 389-391: 1979.
GANCHROW,
SELTZER AND BII(THACH()
36. Luts, A.; Montavon, P.; Lindstrand, K., Sundler, [-. Peptide-conraining nerve fibers in the circumvallate papillae. Reg. Pept. 27:209226: 1990. 37. Massi, M.; Polidori, C.; Perfumi, M.; Ciccocioppo, R.; DeCaro, G.; Bacciarelli, C.; Manzini, S. Effect of capsalcin neonatal treatment on the salt intake of the adult rat. Pharmacol. Biochem. Behav. 40: 163-168, 1991. 38. Mistretta, C. M. Topographical and histological study of the developing rat tongue, palate and taste buds. In: Bosma, J. F., ed. Oral sensation and perception, vol. 3: The mouth of the infant. New York: Thomas; 1972:163-167. 39. Montavon, Ph.; Lindstrand, K.; Luts, A.; Sundler, F. Peptide-containing nerve fibers in the fungiform papillae of pigs and rats. Reg. Pept. 32:141-150; 1991. 40. Nagy, J. I.; Goedert, M.; Hunt, S. P.; Bond, A. The nature of the substance P-containing nerve fibres in taste papillae of the rat tongue. Neuroscience 7:3137-3151; 1982. 41. Nagy, J. 1.; Iverson, L. L.; Goedert, M.; Chapman, D.; Hunt, S. P. Dose-dependent effects of capsaicin on primary sensory neurons in the neonatal rat. J. Neurosci. 3:399-406; 1983. 42. Nishimoto, T.; Akai, M.; Inagaki, S.; Shiosaka, S.; Shimizu, Y.; Yamamoto, K.; Senba, E.; Sakanaka, M.; Takatsuki, K.; Hara, Y.; Takagi, H.; Matsuzaki, T.; Kawai, Y.; Tohyama, M. On the distribution and origins of substance P in the papillae of the tongue: An experimental and immunohistochemical study. J. Comp. Neurol. 207:85-92; 1982. 43. Nishimoto, T.; Ichikawa, H.; Wakisaka, S.; Matsuo, S.; Yamamoto, K.; Nakata, T.; Akai, M. Immunohistochemical observation on substance P in regenerating taste buds of the rat. Anat. Rec. 212: 430-436; 1985. 44. Nowlis. G. H.; Frank, M. Qualities in hamster taste: Behavioral and neural evidence. In: LeMagnen, J.; Macleod, P., eds. Olfaction and taste, vol. 4. London: Information Retrieval, Inc.; 1977:241248. 45. Scadding, J. W. The permanent anatomical effects of neonatal capsaicin on somatosensory nerves. J. Anat. 131:473-484; 1980. 46. Schiffman, S.; Orlandi, M.; Erickson, R. P. Changes in taste and smell with age: Biological aspects. In: Ordy, J. M.; Brizzee, K., eds. Sensory systems and communication in the elderly, vol. 10. Aging. New York: Raven Press; 1979:247-267. 47. Serova, O. N. Changes in activity of the taste receptor apparatus and preference for sodium chloride solution in newborn rats treated with capsaicin. Bull. Exp. Biol. Med. 109:269-271; 1990. 48. Silver, W. L.; Mason, J. R.; Marshall, D. A.; Maruniak, J. A. Rat trigeminal, olfactory and taste responses after capsaicin densensitization. Brain Res. 333:45-54; 1985. 49. Solov'eva, N. A.; Esakov, A. I. Pharmaco-histochemical study of monoamine-containing taste bud cells in capsaicin-induced substance P deficiency. Bull. Exp. Biol. Med. (English Translation). 102:1601 1603; 1986. 50. Valenstein, E. S.; Cox, V. C.; Kakolewski, J. W. Further studies of sex differences in taste preferences with sweet solutions. Psychol. Rep. 20:1231-1234; 1967. 51. Wirtshafter, D.; Asin, K. E. A comment on the practice of expressing food and water intake as proportion of body weight. Physiol. Behav. 28:183-185; 1982. 52. Yamada, T. Chorda tympani responses to gustatory stimuli in developing rats. Jpn. J. Physiol. 30:631-654; 1980. 53. Yamasaki, H.; Kubota, Y.; Takagi, H.; Tohyama, M. Immunoelectronmicroscopic study on the fine structure of substance P-containing fibers in the taste buds of the rat. J. Comp. Neurol. 227:380-392; 1984. 54. Yamasaki, H.; Kubota, Y.; Tohyama, M. Ontogeny of substance-P containing fibers in the taste buds and surrounding epithelium. I. Light microscopic analysis. Dev. Brain. Res. 18: 301-305; 1985.