Selective Deletion of Sodium Salt Taste during Development Leads to Expanded Terminal Fields of Gustatory Nerves in the Adult Mouse Nucleus of the Solitary Tract.

Neuronal activity plays a key role in the development of sensory circuits in the mammalian brain. In the gustatory system, experimental manipulations now exist, through genetic manipulations of specific taste transduction processes, to examine how specific taste qualities (i.e., basic tastes) impact the functional and structural development of gustatory circuits. Here, we used a mouse knock-out model in which the transduction component used to discriminate sodium salts from other taste stimuli was deleted in taste bud cells throughout development. We used this model to test the hypothesis that the lack of activity elicited by sodium salt taste impacts the terminal field organization of nerves that carry taste information from taste buds to the nucleus of the solitary tract (NST) in the medulla. The glossopharyngeal, chorda tympani, and greater superficial petrosal nerves were labeled to examine their terminal fields in adult control mice and in adult mice in which the α-subunit of the epithelial sodium channel was conditionally deleted in taste buds (αENaC knockout). The terminal fields of all three nerves in the NST were up to 2.7 times greater in αENaC knock-out mice compared with the respective field volumes in control mice. The shapes of the fields were similar between the two groups; however, the density and spread of labels were greater in αENaC knock-out mice. Overall, our results show that disruption of the afferent taste signal to sodium salts disrupts the normal age-dependent "pruning" of all terminal fields, which could lead to alterations in sensory coding and taste-related behaviors. SIGNIFICANCE STATEMENT: Neural activity plays a major role in the development of sensory circuits in the mammalian brain. To date, there has been no direct test of whether taste-elicited neural activity has a role in shaping central gustatory circuits. However, recently developed genetic tools now allow an assessment of how specific taste stimuli, in this case sodium salt taste, play a role in the maturation of the terminal fields in the mouse brainstem. We found that the specific deletion of sodium salt taste during development produced terminal fields in adults that were dramatically larger than in control mice, demonstrating for the first time that sodium salt taste-elicited activity is necessary for the normal maturation of gustatory inputs into the brain.

Postnatal development of chorda tympani axons in the rat nucleus of the solitary tract.

The chorda tympani nerve (CT), one of three nerves that convey gustatory information to the nucleus of the solitary tract (NTS), displays terminal field reorganization after postnatal day 15 in the rat. Aiming to gain insight into mechanisms of this phenomenon, CT axon projection field and terminal morphology in NTS subdivisions were examined using tract tracing, light microscopy, and immunoelectron microscopy at four postnatal ages: P15, P25, P35, and adult. The CT axons that innervated NTS rostrolateral subdivision both in the adult and in P15 rats were morphologically distinct from those that innervated the rostrocentral, gustatory subdivision. In both subdivisions, CT terminals reached morphological maturity before P15. Rostrolateral, but not rostrocentral axons, went through substantial axonal branch elimination after P15. Rostrocentral CT synapses, however, redistribute onto postsynaptic targets in the following weeks. CT terminal preference for GABAergic postsynaptic targets was drastically reduced after P15. Furthermore, CT synapses became a smaller component of the total synaptic input to the rostrocentral NTS after P35. The results underlined that CT axons in rostrocentral and rostrolateral subdivisions represent two distinct populations of CT input, displaying different morphological properties and structural reorganization mechanisms during postnatal development.

Chorda tympani nerve terminal field maturation and maintenance is severely altered following changes to gustatory nerve input to the nucleus of the solitary tract.

Neural competition among multiple inputs can affect the refinement and maintenance of terminal fields in sensory systems. In the rat gustatory system, the chorda tympani, greater superficial petrosal, and glossopharyngeal nerves have distinct but overlapping terminal fields in the first central relay, the nucleus of the solitary tract. This overlap is largest at early postnatal ages followed by a significant refinement and pruning of the fields over a 3 week period, suggesting that competitive mechanisms underlie the pruning. Here, we manipulated the putative competitive interactions among the three nerves by sectioning the greater superficial petrosal and glossopharyngeal nerves at postnatal day 15 (P15), P25, or at adulthood, while leaving the chorda tympani nerve intact. The terminal field of the chorda tympani nerve was assessed 35 d following nerve sections, a period before the sectioned nerves functionally regenerated. Regardless of the age when the nerves were cut, the chorda tympani nerve terminal field expanded to a volume four times larger than sham controls. Terminal field density measurements revealed that the expanded terminal field was similar to P15 control rats. Thus, it appears that the chorda tympani nerve terminal field defaults to its early postnatal field size and shape when the nerves with overlapping fields are cut, and this anatomical plasticity is retained into adulthood. These findings not only demonstrate the dramatic and lifelong plasticity in the central gustatory system, but also suggest that corresponding changes in functional and taste-related behaviors will accompany injury-induced changes in brainstem circuits.

The effects of dietary protein restriction on chorda tympani nerve taste responses and terminal field organization.

Prenatal dietary sodium restriction produces profound developmental effects on rat functional taste responses and formation of neural circuits in the brainstem. Converging evidence indicates that the underlying mechanisms for these effects are related to a compromised nutritional state and not to direct stimulus-receptor interactions. We explored whether early malnourishment produces similar functional and structural effects to those seen following dietary sodium restriction by using a protein deficient, sodium replete diet. To determine if early dietary protein-restriction affects the development of the peripheral gustatory system, multi-fiber neurophysiological recordings were made from the chorda tympani nerve and anterograde track tracing of the chorda tympani nerve into the nucleus of the solitary tract (NTS) was accomplished in rats fed a protein-restricted or a control diet (6% and 20%, respectively). The dietary regimens began on embryonic day 7 and continued until rats were used for neurophysiological recordings (postnatal days (P) 35-50) or for chorda tympani terminal field labeling (P40-50). Responses to a concentration series of NaCl, sodium acetate, KCl, and to 0.50 M sucrose, 0.03 M quinine-HCl, and 0.01 N HCl revealed attenuated responses (30-60%) to sodium-specific stimuli in rats fed the 6% protein diet compared with those fed the 20% protein diet. Responses to all other stimuli were similar between groups. Terminal field volumes were nearly twofold larger in protein-restricted rats compared with controls, with the differences located primarily in the dorsal-caudal zone of the terminal field. These results are similar to the results seen previously in rats fed a sodium-restricted diet throughout pre- and postnatal development, suggesting that dietary sodium- and protein-restriction share similar mechanisms in altering gustatory development.

Gustatory terminal field organization and developmental plasticity in the nucleus of the solitary tract revealed through triple-fluorescence labeling.

Early dietary sodium restriction has profound influences on the organization of the gustatory brainstem. However, the anatomical relationships among multiple gustatory nerve inputs have not been examined. Through the use of triple-fluorescence labeling and confocal laser microscopy, terminal fields of the greater superficial petrosal (GSP), chorda tympani (CT), and glossopharyngeal (IX) nerves were visualized concurrently in the nucleus of the solitary tract (NTS) of developmentally sodium-restricted and control rats. Dietary sodium restriction during pre- and postnatal development resulted in a twofold increase in the volume of both the CT and the IX nerve terminal fields but did not affect the volume of the GSP terminal field. In controls, these nerve terminal fields overlapped considerably. The dietary manipulation significantly increased the overlapping zones among terminal fields, resulting in an extension of CT and IX fields past their normal boundaries. The differences in terminal field volumes were exaggerated when expressed relative to the respective NTS volumes. Furthermore, increased terminal field volumes could not be attributed to an increase in the number of afferents because ganglion cell counts did not differ between groups. Taken together, selective increases in terminal field volume and ensuing overlap among terminal fields suggest an increased convergence of these gustatory nerve terminals onto neurons in the NTS. The genesis of such convergence is likely related to disruption of cellular and molecular mechanisms during the development of individual terminal fields, the consequences of which have implications for corresponding functional and behavioral alterations.

Development of the anterior chordal canal.

Resent advances have led to the reexamination of the intraosseous pathway of the chorda tympani a few years ago and they stated that the nerve never enters the mandibular fossa and its exit the skull base in the sphenopetrosal fissure. In our report, 58 temporal bones were investigated after maceration and formalin fixation in order to understand the development of the anterior chordal canal. Our study revealed that the chorda tympani leaves the tympanic cavity through the tympanosquamosal fissure before formation of the anterior chordal canal of Huguier. This canal is situated parallel to and in front of the musculotubal canal and formed by the processus inferior tegminis tympani and the sphenoid bone between the second and fifth years of age. Prior to the age of 2, only the exit of the bony canal exists which is gradually followed by the appearance of a groove in the growing processus inferior tegminis tympani. The borders of the groove elevate and develop to upper and lower plates which lengthen with similar plates of the sphenoid bone, completing the anterior chordal canal by the fifth postnatal year. The entrance of the canal develops above the petrotympanic fissure and similar to the canal itself, it is also completely formed in the fifth year. In case of an incomplete development the anterior chordal canal remains partially opened laterally which might allow the head of the mandibula to effect the chorda tympani mechanically causing Costen's syndrome.

Age-related decrease of the chorda tympani nerve terminal field in the nucleus of the solitary tract is prevented by dietary sodium restriction during development.

Institution of a low-NaCl diet beginning at embryonic day 3 and continued throughout pre- and postnatal development has widespread effects on the neuroanatomical organization of the first gustatory relay in the nucleus of the solitary tract. To determine when these effects are expressed postnatally, the terminal field of the chorda tympani nerve was compared between sodium-restricted and sodium-replete rats at postnatal days 15-17, postnatal days 25-27, postnatal days 35-37, and adults. Total terminal fields were significantly larger in postnatal days 35-37 and adult sodium-restricted rats compared with aged-matched controls. The group-related differences appear related more to a remodeling of the terminal field in the dorsal zone of the terminal field in controls. Specifically, the terminal field volume in the dorsal zone in controls decreased dramatically from postnatal days 25-27 to postnatal days 35-37 and then again from postnatal days 35-37 to adulthood. In contrast, the fields did not change during development in sodium-restricted rats. These findings suggest that remodeling of the chorda tympani field occurs in controls at about the developmental period of taste response maturation. The lack of remodeling in sodium-restricted rats may be explained by a corresponding lack of functional response development to sodium salts. These results also illustrate the specificity and extent of how early dietary manipulations shape the developing brainstem.

Chorda tympani nerve transection at different developmental ages produces differential effects on taste bud volume and papillae morphology in the rat.

Chorda tympani nerve transection (CTX) results in morphological changes to fungiform papillae and associated taste buds. When transection occurs during neonatal development in the rat, the effects on fungiform taste bud and papillae structure are markedly more severe than observed following a comparable surgery in the adult rat. The present study examined the potential "sensitive period" for morphological modifications to tongue epithelium following CTX. Rats received unilateral transection at 65, 30, 25, 20, 15, 10, or 5 days of age. With each descending age at the time of transection, the effects on the structural integrity of fungiform papillae were more severe. Significant losses in total number of taste buds and filiform-like papillae were observed when transection occurred 5-30 days of age. Significant reduction in the number of taste pores was indicated at every age of transection. Another group of rats received chorda tympani transection at 10, 25, or 65 days of age to determine if the time course of taste bud degeneration differed depending on the age of the rat at the time of transection. Taste bud volumes differed significantly from intact sides of the tongue at 2, 8, and 50 days post-transection after CTX at 65 days of age. Volume measurements did not differ 2 days post-transection after CTX at 10 or 25 days of age, but were significantly reduced at the other time points. Findings demonstrate a transitional period throughout development wherein fungiform papillae are highly dependent upon the chorda tympani for maintenance of morphological integrity.

Developmental changes in sugar responses of the chorda tympani nerve in preweanling rats.

To clarify developmental changes in the gustatory system of the rat, integrated taste responses from the chorda tympani (CT) nerve were recorded and analyzed at different postnatal ages. The response magnitude was calculated relative to the response to the standard, 0.1 M NH4Cl. Even at 1 week of age, the CT responded well to all tested 0.1 M chloride salts (NH4Cl, NaCl, LiCl, KCl, RbCl and CsCl). The responses to 0.1 M NaCl and LiCl increased with increasing age of the rat while response magnitudes to KCl, RbCl and CsCl did not change up to 8 weeks. At 1 week, the integrated response pattern was quite similar to that in adult rats for NaCl, HCl and quinine hydrochloride (QHCl). The concentration-response functions for NaCl, HCl, QHCl and sucrose at 2 weeks were essentially the same as those at 8 weeks. These results suggest that taste buds in the 2-week-old rat are functionally mature for the detection of the four basic taste stimuli. The relative magnitude of the responses to the various sugars was smaller at 1 week compared to the adult rat and reached a maximum at weeks 3-4, then decreased gradually with age. Among the six sugars, sucrose was the most effective followed by lactose. From weeks 1-4, the magnitude of the integrated taste response to fructose was smaller than that to lactose except at 3 weeks of age. Maltose, galactose and glucose were less potent stimuli than the other sugars tested. The response magnitude to lactose at 4 weeks had decreased compared to that for the other sugars. Taste responses to the sugars in preweanling and adult rats were not cross-adapted by the individual sugars. These results suggest that after 1 week of age during postnatal development in the rat, taste information from the CT rapidly increases in its importance for feeding behavior.

Neuron/target plasticity in the peripheral gustatory system.

Taste bud volume on the anterior tongue in adult rats is matched by an appropriate number of innervating geniculate ganglion cells. The larger the taste bud, the more geniculate ganglion cells that innervate it. To determine if such a match is perturbed in the regenerated gustatory system under different dietary conditions, taste bud volumes and numbers of innervating neurons were quantified in adult rats after unilateral axotomy of the chorda tympani nerve and/or maintenance on a sodium-restricted diet. The relationship between taste bud size and innervation was eliminated in rats merely fed a sodium-restricted diet; individual taste bud volumes were smaller than predicted by the corresponding number of innervating neurons. Surprisingly, the relationship was disrupted in a similar way on the intact side of the tongue in unilaterally sectioned rats, with no diet-related differences. The mismatch in these groups was due to a decrease in average taste bud volumes and not to a change in numbers of innervating ganglion cells. In contrast, individual taste bud volumes were larger than predicted by the corresponding number of innervating neurons on the regenerated side of the tongue; again, with no diet-related differences. However, the primary variable responsible for disrupting the function on the regenerated side was an approximate 20% decrease in geniculate ganglion cells available to innervate taste buds. Therefore, the neuron/target match in the peripheral gustatory system is susceptible to surgical and/or dietary manipulations that act through multiple mechanisms. This system is ideally suited to model sensory plasticity in adults.

Rearing on basal or high dietary NaCl modifies chorda tympani nerve responses in rats.

Two experiments were conducted to determine the influence of dietary NaCl level on the integrated responses of the chorda tympani (CT) nerve to salt stimulation alone and mixed with the sodium-channel blocker, amiloride hydrochloride. Five groups of adult male rats were reared on regular chow containing either basal 0.1%, intermediate 1.0%, or high 3.0% NaCl from conception to postnatal day (PD) 30 or from conception to adulthood. Adult rats reared from conception to adulthood on basal dietary NaCl demonstrated a reduction in the CT nerve response to NaCl due to a decrease in the amiloride sensitive transduction mechanism. However, the CT nerve responses of adult rats reared on basal dietary NaCl to PD30 and then switched to intermediate dietary NaCl were similar to those of rats reared for a lifetime on intermediate dietary NaCl. Similarly, the CT nerve responses to NaCl in animals reared on high dietary NaCl from conception to PD30 and then switched to an intermediate NaCl diet were comparable to animals reared on intermediate and basal dietary NaCl. However, we found that exposure to high dietary NaCl led to a greater amiloride inhibition of NaCl responses. Thus, there is critical association between dietary NaCl level over two different exposure periods and CT nerve responsiveness to NaCl specifically regarding the degree of amiloride inhibition.

Neuron/target matching between chorda tympani neurons and taste buds during postnatal rat development.

During postnatal development, a relationship is established between the size of individual taste buds and number of innervating neurons. To determine whether rearrangement of neurons that innervate taste buds establishes this relationship, we labeled single taste buds at postnatal day 10 (P10) and again at either P15, P20, or P40 with retrograde fluorescent neuronal tracers. The number of single- and double-labeled geniculate ganglion cells was counted, and the respective taste bud volumes were measured for the three groups of rats. The current study replicates findings from an earlier report demonstrating that the larger the taste bud, the more geniculate ganglion cells that innervate it. This relationship between taste bud size and number of innervating neurons is not apparent until P40, when taste bud size reaches maturity. These findings are extended here by demonstrating that the number of neurons that innervate taste buds at P10, when taste bud size is small and relatively homogeneous, predicts the size that the respective taste bud will become at maturity. Moreover, while there is some neural rearrangement of taste bud innervation from P10 to P40, rearrangement does not impact the relationship between taste bud size and innervating neurons. That is, the neurons that maintain contact with taste buds from P10 through P40 accurately predict the mature taste bud size. Therefore, the size of the mature taste bud is determined by P10 and relates to the number of sensory neurons that innervate it at that age and the number of neurons that maintain contact with it throughout the first 40 days of postnatal development.

[Developmental changing of rat taste responses from chorda tympani nerve].

In order to clarify developmental changing of gustatory system, histological and electrophysiological experiments were performed in the rat. Histological examination on the anterior tongue innervated by chorda tympani nerve showed that the ratio of matured taste buds which possess a taste pore were only 9% of all taste buds observed at 1 week of postnatal age, and 81.3% at 3 weeks of age. Recording integrated responses from the chorda tympani nerve reveals that taste buds with a pore at 1 week of age responded to NaCl, HCl, and quinine-HCl as well as in adult rats, which suggests that these relatively young taste buds are matured functionally for these three stimuli. However, the response magnitudes for various sugars at 1 week of age were smaller compared to those in the adult rat, reached to the maximum at 3 weeks of age, then decreased gradually with age. Also, results from the experiment of cross-adaptation among different sugars, effects of pronase-E treatment of the tongue, analysis of correlation between on- and off-responses to sugars, showed that qualitative changes for sugar responses continues after 3 weeks of age. These results suggest that functional changes occur in the gustatory processing of sugars during postnatal development in the rat chorda tympani nerve.

Developmental decrease in size of peripheral receptive fields of single chorda tympani nerve fibers and relation to increasing NaCl taste sensitivity.

During development in rats, sheep, and humans, the taste system acquires increasing responsiveness to NaCl, compared with a variety of other salts and chemicals. To better understand the neural basis of changes in salt taste responses, we studied receptive field size and response properties of single chorda tympani nerve fibers in fetal, perinatal, and postnatal sheep. Individual fungiform papillae were stimulated electrically with 5 microA anodal current to determine the location and number of papillae in receptive fields. Response characteristics of NH4Cl, NaCl, and KCl were determined for the entire field. Receptive fields were dissected for later histological reconstruction and taste bud identification. Median receptive field size decreased during development. Field sizes in lambs were smaller than those in younger animals. This decrease was accompanied by an increase in the NaCl/NH4Cl response ratio of single fibers and an increase in the proportion of fibers and associated fields that responded with higher frequency to NaCl, compared with NH4Cl. In addition, for fibers across all age groups, receptive field size correlated negatively with the NaCl/NH4Cl response ratio; that is, fields most responsive to NaCl had fewer papillae than those most responsive to NH4Cl. For all fibers, receptive field size correlated with response frequencies to NH4Cl and KCl but not NaCl. For NaCl-best fibers, receptive field size correlated with the response frequencies to all 3 salts. There was no relation between number of taste buds in a single fungiform papilla and the response frequency elicited during electrical stimulation of the papilla.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphology of chorda tympani fiber receptive fields and proposed neural rearrangements during development.

The average number of fungiform papillae in receptive fields of single chorda tympani nerve fibers decreases during development in sheep, and a greater proportion of small receptive fields that are highly responsive to NaCl, compared with NH4Cl, is acquired. To learn whether there also are developmental differences in the number of taste buds within the papillae in mapped receptive fields, we studied the morphology of receptive fields and fungiform papillae, and also counted fibers in the chorda tympani nerve, in fetal, perinatal, and postnatal sheep. Whether defined as the number of fungiform papillae or as the number of taste buds within papillae, receptive fields of chorda fibers decrease developmentally. Initially, however, there is an increase, and subsequently a decrease, in the number of taste buds per field. The differences in field size cannot be attributed to developmental alterations in numbers of fungiform papillae because the total number of papillae on the tongue remains constant. The average number of taste buds per papilla, however, also increases and then decreases, and the increase in perinatal animals is accompanied by the appearance of large, multipored taste buds. Because there is a significant relation between fungiform papilla size and number of taste buds in the papilla, papilla size could be one regulating factor for taste bud number. Furthermore, the number of chorda tympani nerve fibers apparently increases up to perinatal stages and then decreases postnatally, providing another potential regulating factor for the number of taste buds.(ABSTRACT TRUNCATED AT 250 WORDS)

Addition of functional amiloride-sensitive components to the receptor membrane: a possible mechanism for altered taste responses during development.

The developmental increase in response sensitivity of rat peripheral taste afferents to NaCl and LiCl occurs concomitantly with an increase in sensitivity to the sodium ion transport blocker amiloride. Lingual application of amiloride had no effect on chorda tympani nerve taste responses to monochloride salts in early postnatal rats. However, amiloride suppressed NaCl and LiCl responses in proportion to the increased sensitivity to these stimuli during development in postweaning and adult rats without suppressing responses to NH4Cl and KCl.

Myelination in the chorda tympani of the postnatal rat: a quantitative electron microscope study.

We determined the course of myelination of the chorda tympani in rats aged from 4- to 30-days-old, the interval of the most rapid developmental changes in neurophysiological taste responses and behavioral discrimination among chemical stimuli. The overall number of axons in rats aged from 16- to 30-days-old and in mature 120-day-old animals were the same and averaged 1,500. By 30 days, rats had 80% of the total number of myelinated axons observed in adults, but the average thickness of the myelin sheath per neuron and the proportion of the total cross-sectional area that were only about 60% of adult values. Observed increases in myelination closely parallel decreasing response latencies of single chorda tympani fibers to tongue stimulation with salts.

Neural basis of developing salt taste sensation: response changes in fetal, postnatal, and adult sheep.

To learn whether salt taste responses change during mammalian development, we recorded from multifiber preparations of the chorda tympani while stimulating the anterior tongue in sheep fetuses, lambs, and adults. Stimuli were 0.5 M NH4Cl, KCl, NaCl, and LiCl, and 0.05-0.75 M concentration series of the first three salts. Ultrastructural studies were made of taste buds at different ages to determine whether morphological elements such as microvilli and tight junctions are present in young fetuses. Substantial changes occur in relative salt taste responses, throughout development. In fetuses that are beginning the last third of gestation, NaCl and LiCl elicit much smaller response magnitudes than NH4Cl and KCl. Throughout the rest of gestation and postnatally, the NaCl and LiCl responses gradually increase in magnitude relative to NH4Cl and KCl. In adults, NaCl, LiCl, and NH4Cl all elicit similar response magnitudes and KCl is less effective as a taste stimulus. At ages when response ratios for the 0.5 M salts are changing, there are no changes in shapes of the response/concentration functions for individual salts. Furthermore, microvilli are present on taste bud cell apices and tight junctions are found between cells in the youngest fetuses studied. Therefore, initial stimulus-receptor membrane contacts are probably similar to those in adults. Our data suggest that different membrane components interact with the various monochloride salts and that taste receptors contain different proportions of these various membrane components at different developmental stages. Therefore young taste bud cells do not have the same salt response characteristics as mature cells, and a changing neural substrate underlies development of salt taste function, both pre- and postnatally.