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.

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.

Nav2 is necessary for cranial nerve development and blood pressure regulation.

BACKGROUND: All-trans retinoic acid (atRA) is required for nervous system development, including the developing hindbrain region. Neuron navigator 2 (Nav2) was first identified as an atRA-responsive gene in human neuroblastoma cells (retinoic acid-induced in neuroblastoma 1, Rainb1), and is required for atRA-mediated neurite outgrowth. In this paper, we explore the importance of Nav2 in nervous system development and function in vivo. RESULTS: Nav2 hypomorphic homozygous mutants show decreased survival starting at birth. Nav2 mutant embryos show an overall reduction in nerve fiber density, as well as specific defects in cranial nerves IX (glossopharyngeal) and X (vagus). Nav2 hypomorphic mutant adult mice also display a blunted baroreceptor response compared to wild-type controls. CONCLUSIONS: Nav2 functions in mammalian nervous system development, and is required for normal cranial nerve development and blood pressure regulation in the adult.

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.

Immunohistochemical distribution of growth-associated protein 43 (GAP-43) in developing rat nasoincisor papilla.

We employed immunohistochemistry of growth-associated protein 43 (GAP-43) to trace the early development of gustatory nerves and alpha-gustducin to demonstrate mature taste buds in the rat nasoincisor papilla (NP). The sequential changes of gustatory structures revealed eight characteristic stages. One, at embryonic day 16 (E16), GAP-43-immunoreactive (IR) nerve fibers were observed in close relation with presumptive taste buds in the lateral apical epithelium on each side of NP; meanwhile, no immunoreactivity could be observed in the papillary epithelium. Two, at E17, fine GAP-43-IR nerve fibers first invaded the apical epithelium of the papilla. Three, at E19, GAP-43-IR nerve fibers were extensive in apical epithelium and colonized in immature taste buds. Four, at E20, GAP-43-IR nerve fibers were first observed in ductal epithelium (lining the medial wall of nasoincisor ducts). Five, at postnatal day 1 (P1), immunoreactive nerve fibers first coincided with immature taste buds in the ductal epithelium. Six, at P3, alpha-gustducin-IR cells identical for mature taste buds were simultaneously demonstrated in both apical and ductal epithelium. Seven, at P14, progressive taste bud proliferation and maturation as well as neural invasion were demonstrated in all regions of the epithelium. Eight, during advanced stage in adult animals, extensive innervation was traced especially in close relation with taste buds. The sequential topographic patterns of NP gustatory structures seem very specific as compared to those in other locations of mammalian gustatory system. The present study reveals that gustatory nerves preceded the development of taste buds. However, further investigations are required to examine such a characteristic model for the neurogenic theory of taste induction.

Depolarization waves in the embryonic CNS triggered by multiple sensory inputs and spontaneous activity: optical imaging with a voltage-sensitive dye.

Previously, we discovered a novel type of depolarization wave in the embryonic chick brain by using a multiple-site optical recording technique with a fast voltage-sensitive dye. This depolarization wave traveled widely over almost all the region of the CNS. This profile has raised the possibility that the depolarization wave plays some global roles in development of the CNS, rather than contributing to a specific neuronal circuit formation. To obtain more information concerning this issue, in the present study, we examined whether the depolarization wave was triggered by various types of peripheral nerve inputs. Stimulation applied to the vagus, glossopharyngeal, cochlear and trigeminal nerves evoked widely spreading depolarization waves with similar spatiotemporal distribution patterns. The developmental sequence of wave expression was parallel to the development of the excitatory postsynaptic potentials in each sensory nucleus. The depolarization wave was accompanied by a Ca(2+)-wave, suggesting that not only electrical synchrony, but also large-scale Ca(2+)-transients may affect developmental processes in the embryonic brain. Furthermore, we found that the depolarization wave also occurred spontaneously. The waveform and distribution patterns of the spontaneous optical signals were similar to those of the cranial nerve-evoked depolarization wave. These results demonstrated that the depolarization wave in the embryonic chick brain is triggered by multiple sources of external and endogenous activity. This profile supports the idea that this depolarization wave may not serve as a simple regulator of specific neuronal circuit formation, but might play more global roles in CNS development.

Visualization of cranial motor neurons in live transgenic zebrafish expressing green fluorescent protein under the control of the islet-1 promoter/enhancer.

We generated germ line-transmitting transgenic zebrafish that express green fluorescent protein (GFP) in the cranial motor neurons. This was accomplished by fusing GFP sequences to Islet-1 promoter/enhancer sequences that were sufficient for neural-specific expression. The expression of GFP by the motor neurons in the transgenic fish enabled visualization of the cell bodies, main axons, and the peripheral branches within the muscles. GFP-labeled motor neurons could be followed at high resolution for at least up to day four, when most larval neural circuits become functional, and larvae begin to swim and capture prey. Using this line, we analyzed axonal outgrowth by the cranial motor neurons. Furthermore, by selective application of DiI to specific GFP-positive nerve branches, we showed that the two clusters of trigeminal motor neurons in rhombomeres 2 and 3 innervate different peripheral targets. This finding suggests that the trigeminal motor neurons in the two clusters adopt distinct fates. In future experiments, this transgenic line of zebrafish will allow for a genetic analysis of cranial motor neuron development.

Developmental changes in membrane properties of chemoreceptor afferent neurons of the rat petrosal ganglia.

Carotid body chemoreceptors increase their responsiveness to hypoxia in the postnatal period, but the mechanism for this increase is unresolved. The purpose of the present study was to examine developmental changes in cellular characteristics of chemoreceptor afferent neurons in the petrosal ganglia with the underlying hypothesis that developmental changes occur and may account for the developmental increase in chemoreceptor responsiveness. Chemoreceptor complexes (carotid body, sinus nerve, glossopharyngeal nerve, and petrosal ganglia) were harvested from rats, aged 3-40 days, and intracellular recordings were obtained from petrosal ganglion neurons using sharp electrode impalement. All chemoreceptor neurons across ages were C fibers with conduction velocities <1 m/s and generated repetitive action potentials with depolarization. Resting membrane potential was -61.3 +/- 0.9 (SE) mV (n = 78) and input resistance was 108 +/- 6 MOmega and did not significantly change with age. Cell capacitance was 32.4 +/- 1.7 pF and did not change with age. Rheobase averaged 0.21 +/- 0.02 nA and slightly increased with age. Action potentials were followed by an afterhyperpolarization of 12.4 +/- 0.6 mV and time constant 6.9 +/- 0.5 ms; only the time constant decreased with age. These results, obtained in rat, demonstrate electrophysiologic characteristics which differ substantially from that previously described in cat chemoreceptor neurons. In general developmental changes in cell characteristics are small and are unlikely to account for the developmental increase in chemoreceptor responsiveness with age.

Chemoafferent degeneration and carotid body hypoplasia following chronic hyperoxia in newborn rats.

1. To define the role of environmental oxygen in regulating postnatal maturation of the carotid body afferent pathway, light and electron microscopic methods were used to compare chemoafferent neurone survival and carotid body development in newborn rats reared from birth in normoxia (21 % O2) or chronic hyperoxia (60 % O2). 2. Four weeks of chronic hyperoxia resulted in a significant 41 % decrease in the number of unmyelinated axons in the carotid sinus nerve, compared with age-matched normoxic controls. In contrast, the number of myelinated axons was unaffected by hyperoxic exposure. 3. Chemoafferent neurones, located in the glossopharyngeal petrosal ganglion, already exhibited degenerative changes following 1 week of hyperoxia from birth, indicating that even a relatively short hyperoxic exposure was sufficient to derange normal chemoafferent development. In contrast, no such changes were observed in the vagal nodose ganglion, demonstrating that the effect of high oxygen levels was specific to sensory neurones in the carotid body afferent pathway. Moreover, petrosal ganglion neurones were sensitive to hyperoxic exposure only during the early postnatal period. 4. Chemoafferent degeneration in chronically hyperoxic animals was accompanied by marked hypoplasia of the carotid body. In view of previous findings from our laboratory that chemoafferent neurones require trophic support from the carotid body for survival after birth, we propose that chemoafferent degeneration following chronic hyperoxia is due specifically to the loss of target tissue in the carotid body.

Differential taste responses of mouse chorda tympani and glossopharyngeal nerves to sugars and amino acids.

The differential taste responses of the chorda tympani (CT) and the glossopharyngeal (GL) nerves in preweanling and adult mice were examined by comparing magnitudes of responses to six sugars and 10 amino acids. The results indicate that for sugars the responses of the CT nerve are greater than those of the GL nerve while for umami and essential amino acids the responses of the GL nerve are greater than those of the CT nerve. Such differential taste responses of the CT and GL nerves does not prominently change during development.

Tactile and taste sensory maturation of the frog larvae tongue.

The maturation process of organs in the oral cavity of the larvae of bullfrogs, Rana catesbeiana, corresponding to the larval stages XVIII to XXV distinguished by Taylor and Kallros Anat. Rec., 94 (1946) 7-25, were investigated. In this study, these larval stages were divided by the width of the mouth slit into A1, A2, B and C stages. The A1 and A2 stages corresponded to larval stages XVIII-XIX and XX, respectively. The B and C stages corresponded to stages XXI and XXII-XXV, respectively. In stage A1, mechanical and chemical stimulation of the tongue rudiment, in most larvae, failed to elicit afferent discharges in glossopharyngeal nerves. However, in a small number of larvae, obvious afferent discharges of these nerves were generated. In stage A2, glossopharyngeal nerves in most larvae began responding to the same stimulations. The amplitude increased with decreasing duration of afferent discharges, resembling those of the adult. In stage B, the first glossopharyngeal-hypoglossal nerve reflex (G-H reflex) was elicited by mechanical stimulation of the tongue. In stage C, the G-H reflex was also induced by chemical stimulation and the latency of the G-H reflex elicited by mechanical stimulation decreased. In this stage, expansion and extension of the tongue increased. Therefore, the natural function of this reflex for the intake of food may follow this stage.

Artificial rearing alters development of the nucleus of the solitary tract.

Previous studies have shown that damage induced to fungiform papillae of the anterior tongue at postnatal day 2 (P2) alters both pre- and postsynaptic development of gustatory recipient zones within the rostral nucleus of the solitary tract (NST). The present study was conducted to determine whether or not artificial rearing (AR) manipulations, which reduce normal orochemical stimulation during early postnatal development, would be sufficient to produce alterations in anatomical development of the rostral gustatory NST. Two groups of Long-Evans hooded rats were examined. One group received normal rearing with a lactating dam from birth to weaning (mother reared; MR). A second group of animals received artificial rearing via intragastric cannulae between the ages of P4 and P14, and were thereafter returned to lactating dams until the age of weaning (P21). Following weaning and maturation to adulthood (P49), the organization of gustatory afferent terminal fields in the NST was examined using fluorescent tracing procedures which permit the simultaneous visualization of gustatory afferent terminal fields arising from the seventh and ninth cranial nerves. Results show that AR manipulations between the ages of P4 and P14 produce alterations in development of gustatory afferent terminal fields in the NST that are essentially similar to those observed following early postnatal receptor damage. These results confirm previous suggestions that orochemical stimulation during a limited portion of rats' postnatal life is essential in inducing normal presynaptic development in the gustatory NST.

Developmental regulation of tyrosine hydroxylase expression in primary sensory neurons of the rat.

The regulation of transmitter phenotype in primary sensory neurons remains poorly understood. However, recent studies of catecholaminergic (CA) sensory neurons suggest that expression of this particular phenotype may be related to innervation of specific peripheral tissues. In the glossopharyngeal petrosal ganglion (PG) of adult rats, for example, the vast majority of CA sensory neurons innervate a single target, the carotid body. The present study was undertaken, therefore, to begin investigating factors that underlie CA differentiation in sensory neurons, using the rat PG as a model system. Immunocytochemical, biochemical, and morphometric methods were used to investigate the normal time course of CA development in the PG in vivo, employing tyrosine hydroxylase (TH) as a phenotypic marker. These studies revealed two temporally distinct waves of TH expression during embryogenesis. TH immunoreactivity was initially detectable on Embryonic Day (E) 11.5; the number of stained cells increased markedly by E12.5 and then fell off sharply to near 0 by E15.5. Simultaneous immunostaining for TH and neurofilament proteins revealed a high proportion of double-labeled perikarya on E12.5, indicating that the transiently TH-positive cells are neurons. A second, sustained phase of TH expression began on E16.5, and by Postnatal Day 1 adult numbers of TH-containing ganglion cells were present. Western blot analysis demonstrated that TH levels per cell rose 3.5-fold in the perinatal period, indicating that maturation of this particular catecholaminergic trait in PG sensory neurons is highly regulated around birth. Morphometric techniques were used to define the relationship between neurons that transiently exhibit TH immunoreactivity early in gangliogenesis and those that maintain enzyme expression in the mature PG. These studies revealed separate and distinct growth curves for the early and late TH cells, respectively, demonstrating that the appearance, disappearance, and reappearance of immunoreactive cells reflects the differentiation of two separate populations of PG neurons. Moreover, these data indicate that TH expression in the population of CA cells that persists in the mature PG begins around E16.5. This is after peripheral target innervation has begun, raising the possibility that neuron-target interactions regulate biochemical differentiation of these CA sensory neurons.

Proliferation of taste buds in the foliate and vallate papillae of postnatal hamsters.

The growth of the taste system in the hamster is considered in comparison to the postnatal development of other organ systems and the entire animal. No taste buds are present in vallate or foliate papillae of the hamster at birth, but they attain both the appearance and numbers of adult taste buds within 5 weeks of age. The most rapid increase in the number of taste buds occurs within the first 10 days of life, and this proliferation anticipates the weaning of hamsters which occurs by about three weeks of age. Foliate taste buds reach a maximal number within two months, but vallate taste buds continue to increase in number through 4 months of age. Taste bud proliferation and development occur earlier and more rapidly than in other organ systems. This early development of taste buds may protect the weanling hamster against accidental poisoning by noxious plants, and it may also reinforce the food intake which is required for normal growth.

Development of the sensory systems in the larval and metamorphosing European grass frog (Rana temporaria L.).

It was investigated when and how the four examined sensory systems and the behaviour performances depending on them are affected by metamorphosis and whether they develop at an uniform rate from hatching up to metamorphosis. The following conclusions were reached on the basis of the results: -- The central nervous system starts to metamorphose earlier than the habitus and the viscera. -- Motor reactions based on the new capacities of the metamorphosed sensory systems only set in after metamorphosis. -- During metamorphosis the threshold of general sensitivity for the release of motor reactions is drastically raised. -- The sense of taste does not begin to function until after metamorphosis. -- The olfactory sense is the only one of the four tested systems that functions immediately after hatching. -- The level of development of the sensory systems and motor reactions is low in the early larva. The lack of taxis is characteristic of this. -- There is a phase of accelerated development of the sensory systems and motor reactions between the early and late larval stages. -- By comparison with the situation after metamorphosis the sensory systems of the old larva are fairly simple in their structure, but the motor reactions they permit are sufficient for the larva to cope with its aquatic biotope. -- Not until after the morphological structures have attained a new level of development do the relevant motor reactions show a corresponding advance.

Neuroplasticity of primary afferents in the neo-natal cat and some results of early deafferentation of the trigeminal spinal nucleus.

The plasticity of cervical primary afferents has been investigated in young adult cats in which the trigeminal root, together with most of the ganglion, had been excised via a new approach to these structures during the first week after birth. Once degeneration debris had disappeared, bilateral ganglionectomies of the upper three cervical dorsal roots were done in five animals and the IXth and Xth roots transected on the side of the chronic trigeminal denervation in one instance; the degeneration pattern on the chronically denervated side was compared to that on the normal side. There was mild evidence of increased degeneration of the cervical afferents in the C1 segment and in the medulla (subnucleus interpolaris of V) on the chronically denervated side. The proliferation was more apparent in the kitten operated at three days of age and was only vestigial in animals operated at six or seven days; it had no appreciable tendency to extend into the large contiguous pool of denervated trigeminal neurons. There was no evidence of sprouting of the IXth and Xth nerve afferents. These observations indicate that primary afferents of the upper cervical roots and of the glossopharyngeal and vagus nerves have little neuroplastic potential even at early stages. The literature on neuroplasticity is controversial and a brief review of this together with suggestions regarding the reasons for some of these conflicts is presented. Some structural and functional effects of chronic trigeminal denervation are briefly described.