days. There was a concomitant decrease in amiloride sensitivity during this however, functional taste responses seem unaffected.

Transcription

1 J. Physiol. (1987), 393, pp With 6 text-figures Printed in Great Britain SUSCEPTIBILITY OF THE DEVELOPING RAT GUSTATORY SYSTEM TO THE PHYSIOLOGICAL EFFECTS OF DIETARY SODIUM DEPRIVATION BY DAVID L. HILL From the Department of Psychology, University of Virginia, Charlottesville, VA , U.S.A. (Received 7 October 1986) SUMMARY 1. Multifibre responses were recorded from the chorda tympani nerve in rats fed either a NaCl-deficient diet or a NaCl-replete diet from 3 days post-conception to at least 28 days post-natal. Responses were also recorded in rats fed the NaCldeficient diet during early development and then fed the NaCl-replete diet for 1-2 days beginning at 28 days post-natal, and in rats fed the NaCl-deficient diet only as adults. The epithelial sodium transport blocker, amiloride, was used to study the physiological effects of the diet on taste receptor membrane function and to characterize the events involved in recovery of function. 2. Responses to lingual application of sodium salts increased with increasing stimulus concentration; however, response magnitudes were reduced in rats fed the NaCl-deficient diet during early development compared to controls. Responses to non-sodium salts and non-salt stimuli were similar to controls. Amiloride was ineffective in suppressing taste responses to NaCl in deprived rats but effectively suppressed responses in controls by at least 5 %. After early-deprived rats were fed a NaCl-replete diet, responses to sodium salts recovered to control levels within 15 days. There was a concomitant decrease in amiloride sensitivity during this period. 3. Rats fed the NaCl-deficient diet from early gestation through adulthood had responses similar to younger deprived rats in that sodium responses were lower than controls. However, rats deprived only as adults were similar to controls. 4. The peripheral gustatory system in developing rats is susceptible to the sodium content of the diet and is 'plastic' in that early effects can be reversed by restricting dietary sodium. Once dietary manipulations are instituted past a sensitive period, however, functional taste responses seem unaffected. INTRODUCTION The sense of taste matures post-natally in the rat. Responses of peripheral and central taste neurones increase almost fourfold in rats aged 7-5 days post-natal to stimulation of the anterior tongue with salts (Yamada, 198; Hill & Almli, 198; Ferrell, Mistretta & Bradley, 1981; Hill, Mistretta & Bradley, 1982; Hill, Bradley & Mistretta, 1983; Hill, 1987b). Furthermore, action potential frequencies of single

2 414 D. L. HILL chorda tympani fibres to lingual stimulation with NaCl or LiCl increase while responses to NH4Cl remain constant. Therefore, sensitivities to some salt stimuli are mature at 2 weeks post-natal whereas those to NaCl, for example, develop much later. Recent evidence suggests that the developmental increase in sensitivity to NaCl depends on the incorporation of functional membrane components sensitive to the epithelial sodium transport blocker, amiloride (Hill & Bour, 1985; Hill, 1987a). Despite knowledge of these functional changes in the rat gustatory system during normal development, little is known about the environmental determinates of these characteristics. By comparison, work on the visual, auditory and olfactory systems show that the development of normal neurophysiological, anatomical and behavioural characteristics depends on specific features of early sensory stimulation (e.g. Blakemore & Cooper, 197; Hirsch & Spinelli, 197; Hubel & Wiesel, 197; Gottlieb, 1975; Coopersmith, Henderson & Leon, 1986). For instance, either restricted or enhanced stimulation during development can modify sensory function with the most profound effects occurring when instituted during the period of maximal functional change (Wiesel & Hubel, 1963; 1965; Hubel & Wiesel, 197; Hubel, Wiesel & LeVay, 1976; Kerr, Ostapoff & Rubel, 1979). Neural responses from the peripheral gustatory system can also be modified by environmental factors during early development. Responses to sodium salts from the whole chorda tympani nerve are lower in rats fed a salt-free diet during early development compared to controls, whereas responses to non-sodium stimuli are unaffected (Hill, Mistretta & Bradley, 1986). The studies reported here extend work on the susceptibility of the developing gustatory system to dietary manipulation. Early sodium deprivation resulted in a specific decrease in multifibre responses to sodium salts; these effects were reversed by feeding deprived rats a NaCl-replete diet for at least 15 days. The 'plasticity' of the system appears to relate primarily to alterations in taste receptor membrane components sensitive to amiloride. METHODS Deprivation procedures. Sodium deprivation during development was accomplished by feeding pregnant, Sprague-Dawley rats a sodium-deficient diet consisting of 3 % NaCl; (ICN-Nutritional Biochemicals; Cleveland, OH, U.S.A.) from 3 days post-conception until their pups were weaned at 28 days post-natal. Such a dietary restriction results in lowered sodium levels in the dam's milk compared to levels in dams fed an NaCl-replete diet (Ganguli, Smith & Hanson 1969). Pups born to mothers fed the sodium-deficient diet were either weaned to the same diet or to a sodium-replete diet (1- % NaCl) for 1, 5, 1, 15 or 2 days. The number of rats in each of the six preceding groups were 16, 5, 6, 8, 6 and 3, respectively. Control rats (n = 23) were offspring of mothers always fed the sodium-replete diet and were weaned to the replete diet. Distilled water was available ad libitum to all rats throughout the experiment. Sodium deprivation in adulthood was accomplished by feeding the sodium-deficient diet for days to adult rats (+9 days) raised on the sodium-replete diet (n = 8). Therefore, adultdeprived rats were fed the 3 % NaCl diet for a comparable period as the young animals. Sameaged control rats were fed the 1* % diet throughout development (n = 9). Plasma sodium measures. Plasma sodium levels were obtained from rats always fed the sodiumdeficient diet during development, from rats fed the sodium-replete diet for 1, 5, 1, 15 and 2 days after weaning, from rats fed the sodium-deficient diet as adults, and from the respective control rats. Animals were anaesthetized with a surgical dose of urethane ( 9 g/kg body weight, i.p.) and blood was drawn by cardiac puncture. The blood was centrifuged for 5 min and the plasma fraction was drawn and frozen for later analysis of sodium concentration by flame photometry.

3 PLASTICITY OF THE DEVELOPING GUSTATORY SYSTEM Neurophysiological recordings. Rats were anaesthetized with an intraperitoneal injection of urethane (-9 g/kg body weight) and supplementary injections were given as needed to maintain a surgical level of anaesthesia. Animals were then tracheotomized and placed in a non-traumatic head-holder (Erickson, 1966). Body temperature was regulated between 36 and 38 TC by keeping the rat on a water-circulating heating pad. Using a lateral dissection of the head, the left chorda tympani nerve was exposed, cut near its entrance into the tympanic bulla and dissected from underlying tissues. The nerve was desheathed and placed on a platinum electrode with an indifferent electrode in nearby tissues. Multifibre neural activity from the whole nerve was amplified and stored on magnetic tape. For data analysis, the amplified signal was passed through an a.c. to d.c. converter with a time constant of 5 s (Beidler, 1953). The summated electrical activity was displayed on a rectilinear pen recorder. This measure of neural response reflects the sum of single-fibre responses (Beidler, Fishman & Hardiman, 1955; Hill et al. 1982) and is an appropriate measure for studying responses from a large population of taste receptors. Responses were recorded during stimulation of the anterior tongue with a concentration series of NH4Cl, NaCl, sodium acetate and KCl ( 1--5 M), and during stimulation with 1P M-sucrose, *1 N-HCl and 4 M-quinine hydrochloride. All chemicals were dissolved in distilled water and kept at room temperature during experiments. 15 ml of each stimulus were applied to the anterior tongue with a gravity flow system (8 ml/s) for approximately 3 s followed by a distilled water rinse of at least 1 min. The stability of each preparation was monitored by periodic application of -5 M-NH4Cl. Responses were also recorded to a concentration series of NaCl after lingual application of the epithelial sodium transport blocker, amiloride hydrochloride (5 #M); the solvent for NaCl and NH4Cl solutions and the rinse consisted of 5 ItM-amiloride. Amiloride was 415 used to study alteration in salt sensitivity because previous work has indicated that NaCl-deficient diets selectively affect responses to NaCl (Contreras & Frank, 1979; Hill et al. 1987a), it selectively blocks sodium salt responses and not responses to other salt or non-salt stimuli (e.g. Heck, Mierson & DeSimone, 1984), and it has been successfully used to characterize the development of sodium sensitivity in rat (Hill & Bour, 1985; Hill, 1987a). Response magnitudes were measured as the height of the summated response at 2 s after stimulus application. Therefore, only the steady-state portion of the response was measured and not the tactile response component (Doetsch & Erickson, 197). Since it is not valid to compare absolute response magnitudes between animals or groups (Beidler, 1953), ratios for responses to all stimuli were calculated relative to the 5 M-NH4Cl response. The stimulus 5 M-NH4Cl was chosen as the standard because responses of single peripheral taste neurones are similar during development and because transduction mechanisms for this stimulus seem much different than those to NaCl (Hill et at. 1982; Hill & Bour, 1985). RESULTS Survival and body weights All pups born to mothers fed the 3 % NaCl diet survived to ages when neurophysiologial recordings were made. Body weights at 1, 5 and 1 days were similar for deprived and control rats (Fig. 1). In contrast, deprived rats weighed progressively less than controls from 15 to 28 days of age. The weights of deprived rats changed from 85 % of control weights at 15 days to 58 % of control weights at 28 days with differences in body weight being significantly different throughout this period. Responses from rats fed the sodium-deficient diet during early development Ingestion of a 3 % NaCl diet from early gestation through days post-natal had a profound and selective effect on peripheral taste responses. As shown in Figs 2 and 3A, the relative responses to NaCl in rats fed the -3 % diet were smaller than those in control rats for all concentrations. Similarly, the responses to sodium

4 r Control -- 3% diet D. L. HILL cm._ m * o Age (days) Fig. 1. Body weights of deprived and control rats during early post-natal development. Each point shows the mean+ one standard error (t values = ; d.f. = 51; P<1). A.~~~ *3 % diet %44 *4 2s Control *1 * *5 M-NH4CI NaCI concentration (M) Fig. 2. Integrated responses of the chorda tympani nerve to lingual stimulation with -1-5 M-NaCl and NH4C1 in a rat fed the NaCl-deficient diet during early pre- and postnatal ages (top) and in a control rat (bottom). The deprived rat was 33 days old and the control was 32 days old. In rats fed the NaCl-deficient diet, steady-state responses to NaCl, relative to the NH4Cl response, were lower than those in control rats.

5 PLASTICITY OF THE DEVELOPING GUSTATORY SYSTEM 417 A 1- -Control NaCI 1- Control Sodium acetate 1 9 3% diet - -3% NaCI C - Os*8 a -8 T X -6-6 z ~~~~~~~~z e f-4 X i --- u 4.4 gi B C O-4-5 Concentration (M) Concentration (M) D 1- -.Control NH4Cl 1. -.Control KCI X * -3 % diet 4 *---3 % NaCI diet C.8a 8 z z s ll6 46 v-- o O Concentration (M) Concentration (M) Fig. 3. Steady-state chorda tympani responses in sodium-deprived and control rats to lingual stimulation with NaCl (A), sodium acetate (B), NH4Cl (C) and KCl (D). Mean responses, relative to the 5M-NH3Cl response, were lower to sodium salts in deprived rats compared to controls. Responses to NH4Cl and KCl were comparable. The number of deprived and control rats in which responses to NaCl, NH4Cl and KCl were recorded were sixteen and twenty-three, respectively. Responses to sodium acetate were recorded in four deprived and four control rats. Standard errors are shown for each mean. (t values = 3-7-6&9 for NaCl; (d.f. = 37; P < 1); t value = for sodium acetate (d.f. = 6; P < 1). acetate in deprived rats were less than those in control rats for all concentrations (Fig. 3B). In contrast to the sodium salts, responses were similar between deprived (n = 15) and control rats (n = 18) to each concentration of NH4Cl and KCl (P > 1). Furthermore, sucrose, quinine and HCl elicited similar responses in both groups (P > 1). Responses to sucrose, quinine and HCl in deprived rats were 75 % (mean = -23; S.E.M. = -5), 92% (mean = 33; S.E.M. = 5) and 128% (mean = -33; s.e.m. = 7) of controls, respectively. Therefore, neural responses from the chorda tympani nerve showed a selective effect in rats fed the sodium-deficient diet during development. To examine the underlying mechanisms responsible for the suppressed sodium response in deprived rats, responses to a concentration series of NaCl after lingual application of amiloride were compared with responses before amiloride. Amiloride suppressed responses to all concentrations of NaCl by 54% in control rats (Fig. 4). 14 PHY 393

6 418 D. L.HILL A KL kit -3 % diet 2 s Control Before After Before After -1 M -25 M B o-so Control (before amiloride) s-* 3 % NaCI (before amiloride) o-- o Control (after ami loride) U--u -3 % NaCI (after amiloride) at IP) zcl e -2-3 *5 NaCI concentration (M) Fig. 4. A, integrated chorda tympani responses to 1 and -25 M-NaCl from a deprived rat (top) and a control rat (bottom) before and after 5 min lingual application of 5 /SMamiloride hydrochloride. Steady-state responses were less affected by amiloride in the deprived rat than in the control. B, steady-state chorda tympani responses of deprived and control rats to NaCl before and after lingual application of 5,SM-amiloride hydrochloride. Standard errors are shown for each mean. In contrast, application of amiloride suppressed 1 and.5 M-NaCl responses in deprived rats only 17 and 5%, respectively (Fig 4). As a result of the differential suppression, amiloride eliminated the differences in NaCl responses between deprivation groups (Fig. 4B).

7 PLASTICITY OF THE DEVELOPING GUSTATORY SYSTEM 419 Recovery from deprivation Body weight. Early-deprived rats fed the NaCi-replete diet at 28 days post-natal underwent a rapid and significant increase in body weight. Compared to the final weight obtained during deprivation (at 28 days post-natal), rats increased in body weight 19, 75 and 112 % after being fed the NaCl-replete diet for 1, 5 and 1 days, respectively. Moreover, body weights were similar to controls after 1 days access to the NaCl-replete diet (P > 1). Therefore, early body weight alterations resulting from the deprived diet were not permanent (see Fig. 1). Neurophysiological responses. The chorda tympani in rats fed a 3 % NaCl diet from early gestation to 28 days post-natal and then fed the sodium-replete diet gradually increased in sensitivity to sodium salts and by 15 day was as sensitive as controls. Figure 5A shows responses to a concentration range of NaCl in deprived rats ( day recovery) and rats fed the 1. % NaCl diet for 1, 5 and 1 days before recording from the chorda tympani nerve. Rats fed the 1 % NaCl diet for 1, 5 and 1 days had suppressed NaCl responses to all solutions compared to controls (Fig. 5A). Responses in rats fed the 1I% NaCl diet for 1 day were similar to those fed the diet for 1 days. That is, relative responses to NaCl were increased in rats fed a replete diet for only 1 day, albeit less than in controls. Responses in rats fed the 1P % NaCl diet for 15 and 2 days were similar to controls. Figure 5B shows the amount of response suppression to -1, -25 and 5 M-NaCl by amiloride in deprived rats and rats fed the sodium-replete diet for 1, 5 and 1 days, expressed as a percentage of control. As noted earlier (Fig. 4), amiloride suppressed responses less than 18 % in rats fed the 3 % NaCl diet ( day recovery) and suppressed responses approximately 5 % in controls. By comparison, amiloride had its largest effect on rats fed the 1- % NaCl diet for 1 day and successively smaller an effect on rats with increased exposure to the replete diet. For example, the amount of response suppression to 1 M-NaCl by amiloride was 81, 73, 75, 69, 62 and 54 % for rats fed the 1 % diet for 1, 5, 1, 15 and 2 days and controls, respectively (Fig. 5B). Responses from adult rats always fed the sodium-deficient diet Since responses from rats deprived of NaCl during early development are similar to young 'normal' rats (Hill & Bour, 1985), it was important to demonstrate that the influence of deprivation on the gustatory system was not merely due to delayed development. Therefore, early-deprived male rats (n = 4) were allowed to reach maturity by maintaining them on the 3 % NaCl diet through at least 12 days post-natal before recording from the chorda tympani. At 12 days post-natal, rats always fed the -3 % diet continued to manifest smaller responses to NaCl (Fig. 6). As in younger, deprived rats, adults always fed the NaCl-deficient diet were relatively insensitive to amiloride. For example, amiloride only suppressed responses to -1 M-NaCl by 11-6 % (s.e.m. = 4). Thus, peripheral gustatory alterations were apparent in rats deprived of NaCl from early gestation through adulthood. Finally, responses to sodium salts were similar to controls when rats deprived to adulthood were fed the NaCl-replete diet for at least 15 days (P > -1), demonstrating that the gustatory system remained 'plastic' even though deprivation occurred from early gestational ages through adulthood. 14-2

8 42 D. L.HILL XI day recovery CO 1 day recovery 8 OU 6 4 AL *5 NaCI concentration (m) ~~~~~~~o 175 day recovery I5 as 1 day recovery 15 U 5 day recovery oiis 1 day recovery ~~ cl NaCI concentration (M) Fig. 5. A, responses to 5, 1,.25 and 5 M-NaCl expressed relative to controls in rats fed the NaCl-deficient diet through 28 days post-natal and then fed the NaCl-replete diet for (n = 16, open bars), 1 (n = 5, horizontally hatched bars) 5 (n = 6, filled bars) or 1 days (n= 8, diagonally hatched bars). Response magnitudes of all four groups were significantly less than controls (n = 2) (F(6,6) = ; Tukey post-test, P < 5). B, suppression of 1, -25 and 5 M-NaCl responses expressed relative to control suppression (n = 2) by lingual application of 5 lpm-amiloride hydrochloride in rats intially fed the NaCl-deficient diet during early development and then fed the replete diet for (n = 9, open bars), 1 (horizontally hatched bars), 5 (n = 5, filled bars), or 1 days (n = 8, diagonally hatched bars). Suppression ratios were calculated by dividing the steady-state response after amiloride by the response before amiloride. Vertical lines shown above each bar are the standard errors of the means calculated as a percentage of the control mean. Responses from rats fed the sodium-deficient diet as adults Although dietary NaCl deprivation during early development affected chorda tympani responses to sodium salts, the same effects did not occur in rats fed the 3 % NaCl diet as adults. Response magnitudes to sodium salts in adult rats fed the 3 % NaCl diet for at least 49 days were similar to control rats for all concentrations.

9 PLASTICITY OF THE DEVELOPING GUSTATORY SYSTEM Moreover, responses from the mothers of deprived pups (n = 4) were similar to controls (Fig. 6). This finding further indicates that deprivation must occur early in development in order to produce peripheral gustatory changes because the mothers were deprived for comparable periods as their pups and also had nutritional and electrolyte stores challenged during pregnancy and lactation (Ganguli et al. 1969)., 14 O Mothers 12 i Adult Long-term deprived deprived c4 CL NaCI concentration (M) Fig. 6. Responses to -5, -1, -25 and -5 M-NaCl expressed relative to adult controls (n = 7) from mothers of deprived rats (n = 4, open bars), rats deprived of NaCi as adults (n = 8, horizontally hatched bars) and rats deprived from early development through adulthood (n = 4, filled bars). Only responses from rats continually fed the NaCl-deficient diet during development were affected (t values = , d.f. = 9, P < -1). Vertical lines shown above each bar are the standard errors of the means calculated as a percentage of the control mean. TABLE 1. Plasma sodium levels of control, deprived and 'recovering' rats Mean plasma Deprivation group sodium (mequiv/l) S.E.M n Control day recovery day recovery 131-4* day recovery 132-* day recovery 132-* day recovery day recovery * Significantly greater than controls (P < -5). Plasma sodium measures Plasma sodium levels of rats fed the -3 % NaCl diet during early development ( day recovery) and during adulthood were similar to control rats (P > -1). However, rats initially fed the -3 % diet to 28 days post-natal and then fed the NaCl-replete diet for 1, 5 and 1 days had elevated plasma sodium levels compared to controls (Table 1). Rats fed the replete diet for 15 and 2 days were similar to controls (P > -1; Table 1). Therefore, animals fed the NaCl-replete diet after prolonged deprivation were able to regulate plasma sodium as did controls, whereas animals placed on the replete diet for 1 days or less had greater than control levels. 421

10 422 D. L. HILL DISCUSSION Neurophysiological taste responses from the chorda tympani nerve to sodium salts were decreased in rats fed a sodium-deficient diet consisting of 3 % NaCl during early pre- and post-natal periods compared to control rats fed a NaCl-replete diet containing 1 % NaCl. However, the taste response deficit was selective since responses to non-sodium salts and non-salt stimuli were similar to control rats. This may be due to a reduction in functional amiloride-sensitive sodium components on taste receptor membranes in deprived rats. Amiloride was ineffective in suppressing responses to NaCl in deprived rats but reduced sodium responses in control rats to deprived levels. Thus, amiloride eliminated the differences between sodium-induced activity in the peripheral gustatory system of deprived and control rats. Although there were clear alterations in peripheral gustatory function resulting from early NaCl deprivation, they were not permanent. Chorda tympani responses to NaCl increased to control levels after deprived rats were fed the NaCl-replete diet for at least 15 days. The recovery of function was accompanied by changes in amiloride sensitivity, suggesting that functional amiloride-sensitive membrane components were restored to the taste membrane. Within the first 24 h following ingestion of the NaCl-replete diet, amiloride almost totally suppressed the NaCl response. In fact, amiloride was more effective in suppressing NaCl responses within the first 24 h after feeding the NaCl-replete diet to deprived rats than it was in suppressing responses in controls. A progressive attenuation of the amiloride effect to control levels occurred within 15 days after changing diets. In contrast to the 'plasticity' induced by factors during early development, rats initially fed the NaCl-deficient diet as adults showed no alteration in multifibre chorda tympani nerve responses. Therefore, NaCl deprivation during early development has dramatic and specific effects on peripheral taste responses that cannot be produced by similar procedures initiated after a 'sensitive' period. The changes in sodium and amiloride sensitivity may be due to the effects on the development of the receptor membranes, the synapses between receptor cells and chorda tympani fibres, or chorda tympani fibres themselves. From the current data, it seems likely that the effects relate primarily to alterations in taste receptor membranes. Responses to non-sodium salts or non-salt stimuli were normal, indicating that the general function of the chorda tympani was not altered in deprived rats. Moreover, the rapid 'recovery' in sodium and amiloride sensitivities in deprived rats fed the NaCl-replete diet cannot plausibly be accounted for by mechanisms other than those involving rapid changes in the composition of membrane components sensitive to sodium. Correlative single-fibre experiments will provide insight into the underlying mechanisms. Comparison with normal development The effect of early NaCl deprivation on chorda tympani function parallels the development of chorda tympani function in immature rats. Young rats (12-13 days) have low relative taste responses to NaCl that are insensitive to amiloride (Hill & Bour, 1985). As the normal animal matures, a concomitant increase in NaCl and amiloride sensitivity occurs (Hill & Bour, 1985). The formation of functional

11 PLASTICITY OF THE DEVELOPING GUSTATORY SYSTEM amiloride-sensitive components seems the likely factor in the increase in peripheral gustatory sensitivity to NaCl during normal development and in rats 'recovering' from early NaCl deprivation. The similarities between normal development and recovery following deprivation are not perfect, however, because amiloride-sensitive components apparently increase gradually during normal development whereas they seem to form rapidly (24 h) in recovering animals and then gradually decrease with additional exposure to the replete diet. It is possible that such components can form much faster in recovering rats than during normal development. Alternatively, functional components may form de novo during normal development as new receptor cells are added (Beidler & Smallman, 1965), whereas non-functional components may form during sodium deprivation and become functional only when the replete diet is ingested. The role of circulating factors in recovery Although the identity of factors important in formation of amiloride-sensitive components on taste receptors is not known, it is interesting to note that 'recovered' rats exhibit unusually high plasma sodium levels after changing diets from 3 to 1- % NaCl at 28 days post-natal. This is also the period when the greatest sensitivity to amiloride occurs. It is unlikely that plasma sodium levels per se regulate amiloridesensitive membrane components since rats always fed the NaCl-deficient diet are insensitive to amiloride, yet have similar plasma sodium levels to controls. Furthermore, it seems paradoxical that deprived rats have decreased responses to sodium salts. Factors such as aldosterone that have been demonstrated to induce formation of membrane components sensitive to amiloride should be high in deprived rats and thus result in higher instead of lower relative responses to sodium salts (Will, Lebowitz & Hopfer, 198; Will, DeLisle, Cortright & Hopfer, 1981). However, the effects of circulating factors on formation of sodium-sensitive membrane components vary depending on the species and tissues studied (Crabbe, 198; Palmer, Li, Lindemann & Edelman, 1982). I thank Dr P. C. Brunjes and Mr P. Przekop for critical comments on an earlier draft of the manuscript. This research was supported by National Institutes of Health Grant NS2538 and Research Career Development Award NS964. I also thank Mr Walter Gall of Merck, Sharp and Dohme Research Laboratories for the generous gift of amiloride. 423 REFERENCES BEIDLER, L. M. (1953). Properties of chemoreceptors of tongue of rat. Journal of Neurophysiology 16, BEIDLER, L. M., FISHMAN, I. Y. & HARDIMAN, C. W. (1955). Species differences in taste response. American Journal of Physiology 181, BEIDLER, L. M. & SMALLMAN, R. L. (1985). Renewal of cells within taste buds. Journal of Cell Biology 27, BLAKEMORE, G. & COOPER, G. F. (197). Development of the brain depends on the visual environment. Nature 228, CONTRERAS, R. J. & FRANK, M. E. (1979). Sodium deprivation alters neural responses to gustatory stimuli. Journal of General Physiology 73, COOPERSMITH, R., HENDERSON, S. R. & LEON, M. (1986). Odor specificity of the enhanced neural response following early odor experience in rats. Developmental Brain Research 27,

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