Histoarchitectonics of the cholinergic innervation of the frog tongue

Acetylcholinesterase (AChE) activity was studied in dorsal tongue surface structures and in both tongue nerves (hypoglossal and glossopharyngeal) of frogs (Rana temporaria). AChE was found in nerve fibers of fungiform and filiform papillae, blood vessels, glandular ducts of tongue mucosa, both nerve fibers and also in the bodies of cholinergic neurons in subepithelial connective tissue and along the glossopharyngeal nerve. Their parasympathetic origin was suggested. The experiments with butirilthiocholin have revealed no activity of non-specific cholinesterases in the above structures. Possible role of cholinergic system in the regulation of tongue receptor function is discussed.     

Labeling afferent nerves in the tongue by peripheral transganglionic transport of HRP and WGA-HRP in the rat

Peripheral transganglionic transport of horseradish pcroxidase(HRP) and wheat germ agglutinin–horseradish peroxidaseconjugate (WGA–HRP) was used to label afferent fibersin the taste buds and lingual epithelium of the rat. Microinjectionsof the tracer were made in the brain stem central projectionarea of the afferent nerves to the tongue. Optimal labelingof nerve endings in the tongue was obtained when 2 µlof 20% HRP was injected into the brain stem and postinjectionsurvival times of 24–36 h were used. The distributionof single nerves was studied by using this tracing procedurein combination with strategic transections of the various afferentnerves supplying the tongue. Labeled nerve fibers from the combinedchorda tympani–lingual nerve were found in the epitheliumand in taste buds in the fungiform and anterior foliate papillaeof the anterior 3/4 of the tongue. Labeled nerve fibers in theepithelium of the anterior 2/3 of the tongue but none in tastebuds were found when the lingual nerve alone was studied, althoughnumerous perigeminal fibers were found. The glossopharyngealnerve was found to innervate die posterior 1/4 of the tongueepithelium including the taste buds of the circumvallate papillae.The glossopharyngeal nerve on one side was found to innervatethe taste buds on both sides of the midline. The results showthat this tracing procedure can be a useful supplement to othermethods for studying afferent nerves in the tongue.     

Morphofunctional state of the cholinergic innervation of the rat pancreas during vagotomy]

Cholinergic innervation of the albino rat pancreas was investigated under normal conditions and after bilateral subdiaphragmatic vagotomy (one, three, seven, twenty one days and two months after the operation). In control animals cholinergic nerve fibers were found in all the organ structures -- both in the vascular and in the gland-duct systems. Vagotomy resulted in a short-term increase in the nerve fibers revealed at the end of the first day, followed by their decrease on the seventh day; but in two months the number of detectable nerve fibers approached the initial level.     

The origin of slow potentials on the tongue surface induced by frog glossopharyngeal efferent fiber stimulation

When the glossopharyngeal (GP) nerve of the frog was stimulated electrically, electropositive slow potentials were recorded from the tongue surface and depolarizing slow potentials from taste cells in the fungiform papillae. The amplitude of the slow potentials was stimulus strength- and the frequency-dependent. Generation of the slow potentials was not related to antidromic activity of myelinated afferent fibers in the GP nerve, but to orthodromic activity of autonomic post-ganglionic C fibers in the GP nerve. Intravenous injection of atropine abolished the positive and depolarizing slow potentials evoked by GP nerve stimulation, suggesting that the slow potentials were induced by the activity of parasympathetic post-ganglionic fibers. The amplitude and polarity of the slow potentials depended on the concentration of adapting NaCl solutions applied to the tongue surface. These results suggest that the slow potentials recorded from the tongue surface and taste cells are due to the liquid junction potential generated between saliva secreted from the lingual glands by GP nerve stimulation and the adapting solution on the tongue surface.     

Time course of morphological alterations of fungiform papillae and taste buds following chorda tympani transection in neonatal rats

The time course of structural changes in fungiform papillae was analyzed in rats that received unilateral chorda tympani nerve transection at 10 days of age. Morphological differences between intact and denervated sides of the tongue were first observed at 8 days postsection, with an increase in the number of fungiform papillae that did not have a pore. In addition, the first papilla with a filiform-like appearance was noted on the denervated side at 8 days postsectioning. By 11 days after surgery, the total number of papillae and the number of papillae with a pore were significantly lower on the transected side of the tongue as compared to the intact side. At 50 days postsection, there was an average of 70.5 fungiform papillae on the intact side and a mean of only 20.8 fungiform papillae the denervated side. Of those few remaining papillae on the cut side, an average of 13.5 papillae were categorized as filiform-like, while no filiform-like papillae occurred on the intact side. Significant reduction in taste bud volume was noted at 4 days posttransection and further decrements in taste bud volume were noted at 8 and 30 days postsection. Electron microscopy of the lingual branch of the trigeminal nerve from adult rats that received neonatal chorda tympani transection showed normal numbers of both myelinated and unmyelinated fibers. Thus, in addition to the well-characterized dependence of taste bud maintenance on the chorda tympani nerve, the present study shows an additional role of the chorda tympani nerve in papilla maintenance during early postnatal development.     

Prion infection of oral and nasal mucosa

Centrifugal spread of the prion agent to peripheral tissues is postulated to occur by axonal transport along nerve fibers. This study investigated the distribution of the pathological isoform of the protein (PrP(Sc)) in the tongues and nasal cavities of hamsters following intracerebral inoculation of the HY strain of the transmissible mink encephalopathy (TME) agent. We report that PrP(Sc) deposition was found in the lamina propria, taste buds, and stratified squamous epithelium of fungiform papillae in the tongue, as well as in skeletal muscle cells. Using laser scanning confocal microscopy, PrP(Sc) was localized to nerve fibers in each of these structures in the tongue, neuroepithelial taste cells of the taste bud, and, possibly, epithelial cells. This PrP(Sc) distribution was consistent with a spread of HY TME agent along both somatosensory and gustatory cranial nerves to the tongue and suggests subsequent synaptic spread to taste cells and epithelial cells via peripheral synapses. In the nasal cavity, PrP(Sc) accumulation was found in the olfactory and vomeronasal epithelium, where its location was consistent with a distribution in cell bodies and apical dendrites of the sensory neurons. Prion spread to these sites is consistent with transport via the olfactory nerve fibers that descend from the olfactory bulb. Our data suggest that epithelial cells, neuroepithelial taste cells, or olfactory sensory neurons at chemosensory mucosal surfaces, which undergo normal turnover, infected with the prion agent could be shed and play a role in the horizontal transmission of animal prion diseases.     

Taste cell responses in the frog are modulated by parasympathetic efferent nerve fibers

We studied the anatomical properties of parasympathetic postganglionic neurons in the frog tongue and their modulatory effects on taste cell responses. Most of the parasympathetic ganglion cell bodies in the tongue were found in extremely small nerve bundles running near the fungiform papillae, which originate from the lingual branches of the glossopharyngeal (GP) nerve. The density of parasympathetic postganglionic neurons in the tongue was 8000-11,000/mm(3) of the extremely small nerve bundle. The mean major axis of parasympathetic ganglion cell bodies was 21 microm, and the mean length of parasympathetic postganglionic neurons was 1.45 mm. Electrical stimulation at 30 Hz of either the GP nerve or the papillary nerve produced slow hyperpolarizing potentials (HPs) in taste cells. After nicotinic acetyl choline receptors on the parasympathetic ganglion cells in the tongue had been blocked by intravenous (i.v.) injection of D-tubocurarine (1 mg/kg), stimulation of the GP nerve did not induce any slow HPs in taste cells but that of the papillary nerve did. A further i.v. injection of a substance P NK-1 antagonist, L-703,606, blocked the slow HPs induced by the papillary nerve stimulation. This suggests that the parasympathetic postganglionic efferent fibers innervate taste cells and are related to a generation of the slow HPs and that substance P is released from the parasympathetic postganglionic axon terminals. When the resting membrane potential of a taste cell was hyperpolarized by a prolonged slow HP, the gustatory receptor potentials for NaCl and sugar stimuli were enhanced in amplitude, but those for quinine-HCl and acetic acid stimuli remained unchanged. It is concluded that frog taste cell responses are modulated by activities of parasympathetic postganglionic efferent fibers innervating these cells.     

Tonic activity of parasympathetic efferent nerve fibers hyperpolarizes the resting membrane potential of frog taste cells

We investigated the relationship between the membrane potential of frog taste cells in the fungiform papillae and the tonic discharge of parasympathetic efferent fibers in the glossopharyngeal (GP) nerve. When the parasympathetic preganglionic fibers in the GP nerve were kept intact, the mean membrane potential of Ringer-adapted taste cells was -40 mV but decreased to -31 mV after transecting the preganglionic fibers in the GP nerve and crushing the postganglionic fibers in the papillary nerve. The same result occurred after blocking the nicotinic acetylcholine receptors on parasympathetic ganglion cells in the tongue and blocking the substance P neurokinin-1 (NK-1) receptors in the gustatory efferent synapses. This indicates that the parasympathetic nerve (PSN) hyperpolarizes the membrane potential of frog taste cells by -9 mV. Repetitive stimulation of a transected GP nerve revealed that a -9-mV hyperpolarization of taste cells maintained under the intact GP nerve derives from an approximately 10-Hz discharge of the PSN efferent fibers. The mean frequency of tonic discharges extracellularly recorded from PSN efferent fibers of the taste disks was 9.1 impulses/s. We conclude that the resting membrane potential of frog taste cells is continuously hyperpolarized by on average -9 mV by an approximately 10-Hz tonic discharge from the parasympathetic preganglionic neurons in the medulla oblongata.     

Ethanol modulates the VR-1 variant amiloride-insensitive salt taste receptor. I. Effect on TRC volume and Na+ flux

The effect of ethanol on the amiloride- and benzamil (Bz)-insensitive salt taste receptor was investigated by the measurement of intracellular Na(+) activity ([Na(+)](i)) in polarized rat fungiform taste receptor cells (TRCs) using fluorescence imaging and by chorda tympani (CT) taste nerve recordings. CT responses were monitored during lingual stimulation with ethanol solutions containing NaCl or KCl. CT responses were recorded in the presence of Bz (a specific blocker of the epithelial Na(+) channel [ENaC]) or the vanilloid receptor-1 (VR-1) antagonists capsazepine or SB-366791, which also block the Bz-insensitive salt taste receptor, a VR-1 variant. CT responses were recorded at 23 degrees C or 42 degrees C (a temperature at which the VR-1 variant salt taste receptor activity is maximally enhanced). In the absence of permeable cations, ethanol induced a transient decrease in TRC volume, and stimulating the tongue with ethanol solutions without added salt elicited only transient phasic CT responses that were insensitive to elevated temperature or SB-366791. Preshrinking TRCs in vivo with hypertonic mannitol (0.5 M) attenuated the magnitude of the phasic CT response, indicating that in the absence of mineral salts, transient phasic CT responses are related to the ethanol-induced osmotic shrinkage of TRCs. In the presence of mineral salts, ethanol increased the Bz-insensitive apical cation flux in TRCs without a change in cell volume, increased transepithelial electrical resistance across the tongue, and elicited CT responses that were similar to salt responses, consisting of both a transient phasic component and a sustained tonic component. Ethanol increased the Bz-insensitive NaCl CT response. This effect was further enhanced by elevating the temperature from 23 degrees C to 42 degrees C, and was blocked by SB-366791. We conclude that in the presence of mineral salts, ethanol modulates the Bz-insensitive VR-1 variant salt taste receptor.     

Effects of antidromic activity in gustatory nerve fibers on taste disc cells of the frog tongue

Summary Antidromic electrical stimulation of the lingual branch of the glossopharyngeal (IX) nerve of the frog was carried out while recording intracellular potentials of taste disc cells.Antidromic activation of sensory fibers resulted in depolarization of cells of the upper layer of the disc and most commonly in hyperpolarization of the cells in the lower layer. These changes in potential exhibited latencies greater than 1 s (Fig. 3), and thus cannot be due to electrotonic effects of action potentials in terminals of IX nerve fibers, which have much shorter conduction times. These cell potentials also showed summation, adaptation and post-stimulus rebound (Figs. 3, 4).Depending upon the chemical stimulus used, antidromic activity produced either depression or enhancement of gustatory fiber discharge in response to taste stimuli (Fig. 5).Alteration of the resting membrane potential by current injection did not significantly modify the antidromically evoked potentials (Fig. 8), whereas chemical stimulation of the tongue did (Fig. 7), indicating that these potential changes are not the result of passive electrical processes.These experimental results indicate that the membrane potential of taste disc cells can be modified by antidromic activity in their afferent nerves. This mechanism may be responsible for peripheral interactions among gustatory units of the frog tongue.The research was supported in part by NIH grant NS-09168.     

Dual innervation of arteries and arterioles

Summary Specific histochemical techniques for the demonstration of acetylcholinesterase and of norepinephrine have been used to study the distribution of cholinergic and adrenergic nerve fibers to arteries and arterioles in various organs of cats and dogs, including the male genital apparatus, tongue, skeletal muscle, heart and gastrointestinal tract. Arteries and arterioles in all of these organs showed both cholinergic and adrenergic nerve fibers, although the relative number of each of the types of fiber was variable. The findings provide morphologic evidence for a widespread and generalized dual adrenergic and cholinergic innervation of arteries and arterioles.Supported in part by Grant No. HE 10465 from the USPHS and by a grant from the Monroe County Heart Chapter.     

Taste-responsive neurons in the nucleus of the solitary tract receive gustatory information from both sides of the tongue in the hamster

Taste receptors on the left and right sides of the anterior tongue are innervated by chorda tympani (CT) fibers, which carry taste information to the ipsilateral nucleus of the solitary tract (NST). Although the anterior tongue is essential for taste, patients with unilateral CT nerve damage often report no subjective change in their taste experience. The standing theory that explains the taste constancy is the "release of inhibition", which hypothesizes that within the NST there are inhibitory interactions between inputs from the CT and glossopharyngeal nerves and that the loss of taste information from the CT is compensated by a release of inhibition on the glossopharyngeal nerve input. However, the possibility of compensation by taste input from the other side of the tongue has never been investigated in rodents. We recorded from 95 taste-responsive neurons in the NST and examined their responsiveness to stimulation of the contralateral CT. Forty-six cells were activated, mostly with excitatory responses (42 cells). Activation of NST cells induced by contralateral CT stimulation was blocked by microinjection of lidocaine into the contralateral NST but was not affected by anesthetization of the contralateral parabrachial nuclei (PbN). In addition, the NST cells that were activated by contralateral CT stimulation showed reduced responsiveness to taste stimulation after microinjection of lidocaine into the contralateral NST. These results demonstrate that nearly half of the taste neurons in the NST receive gustatory information from both sides of the tongue. This "cross talk" between bilateral NST may also contribute to the "taste constancy".     

Effects of chorda tympani nerve anesthesia on taste responses in the NST

Human clinical and psychophysical observations suggest that the taste system is able to compensate for losses in peripheral nerve input, since patients do not commonly report decrements in whole mouth taste following chorda tympani nerve damage or anesthesia. Indeed, neurophysiological data from the rat nucleus of the solitary tract (NST) suggests that a release of inhibition (disinhibition) may occur centrally following chorda tympani nerve anesthesia. Our purpose was to study this possibility further. We recorded from 59 multi- and single- unit taste-responsive sites in the rat NST before, during and after recovery from chorda tympani nerve anesthesia. During anesthesia, average anterior tongue responses were eliminated but no compensatory increases in palatal or posterior tongue responses were observed. However, six individual sites displayed increased taste responsiveness during anesthesia. The average increase was 32.9%. Therefore, disinhibition of taste responses was observed, but infrequently and to a small degree in the NST At a subset of sites, chorda tympani-mediated responses decreased while greater superficial petrosal-mediated responses remained the same during anesthesia. Since this effect was accompanied by a decrease in spontaneous activity, we propose that taste compensation may result in part by a change in signal-to-noise ratio at a subset of sites.      

Ethanol modulates the VR-1 variant amiloride-insensitive salt taste receptor. II. Effect on chorda tympani salt responses

The effect of ethanol on the amiloride- and benzamil (Bz)-insensitive salt taste receptor was investigated by direct measurement of intracellular Na(+) activity ([Na(+)](i)) using fluorescence imaging in polarized fungiform taste receptor cells (TRCs) and by chorda tympani (CT) taste nerve recordings. CT responses to KCl and NaCl were recorded in Sprague-Dawley rats, and in wild-type (WT) and vanilloid receptor-1 (VR-1) knockout mice (KO). CT responses were monitored in the presence of Bz, a specific blocker of the epithelial Na(+) channel (ENaC). CT responses were also recorded in the presence of agonists (resiniferatoxin and elevated temperature) and antagonists (capsazepine and SB-366791) of VR-1 that similarly modulate the Bz-insensitive VR-1 variant salt taste receptor. In the absence of mineral salts, ethanol induced a transient decrease in TRC volume and elicited only transient phasic CT responses. In the presence of mineral salts, ethanol increased the apical cation flux in TRCs without a change in volume, increased transepithelial electrical resistance across the tongue, and elicited CT responses that were similar to salt responses, consisting of both a phasic component and a sustained tonic component. At concentrations <50%, ethanol enhanced responses to KCl and NaCl, while at ethanol concentrations >50%, those CT responses were inhibited. Resiniferatoxin and elevated temperature increased the sensitivity of the CT response to ethanol in salt-containing media, and SB-366791 inhibited the effect of ethanol, resiniferatoxin, and elevated temperature on the CT responses to mineral salts. VR-1 KO mice demonstrated no Bz-insensitive CT response to NaCl and no sensitivity to ethanol. We conclude that ethanol increases salt taste sensitivity by its direct action on the Bz-insensitive VR-1 variant salt taste receptor.     

The state of adrenergic and cholinergic fibers in the heart of rabbits subjected to sensitization and anaphylactic shock

It was determined by neurohistochemical methods that after sensitization and anaphylactic shock the adrenergic and cholinergic nervous structures of the heart change. The lowered fluorescence intensity, alterations in density of the adrenergic nervous structures of the heart are observed following the fourth sensitizing injection of normal horse serum. These changes are more obvious on day 7 after anaphylactic shock. The changes in the structure of cholinergic nerve fibers were noted only on day 7 after anaphylactic shock. They manifested in lowered activity of histochemical reaction of nerve elements to acetylcholine in all the regions of the heart.     

Lingual taste buds following application of colchicine to the glossopharyngeal nerve in the rat

Dissection of the glossopharyngeal nerve and application to it of colchicine that blocks axoplasmic drug transport were performed to study the effect of the nerves on the taste buds of foliate lingual papillae. It was observed that colchicine application to the nerve gave rise to destruction of the taste buds. The process of destruction proceeded more slowly as compared to that induced by nerve dissection. Colchicine application led to changes in the protein spectrum of the epithelium of foliate papillae. The absence of changes in the protein spectrum of the epithelium of foliate papillae and the presence of nerve fibers in the epithelium of the taste buds on exposure to colchicine provide evidence against its direct toxic effect on the taste buds, giving rise to their destruction. The changes seen in the taste buds result from the blocked transport of factors that participate in neurotropic control of the taste buds.     

Calbindin D28k-like immunoreactivity in the developing and regenerating circumvallate papilla of the rat

The distribution of calbindin D28k (CB)-like immunoreactivity (-LI) in the circumvallate papilla (CVP) was examined during development and regeneration following bilateral crush injury to the glossopharyngeal nerve in the rat. In the adult CVP, CB-like immunoreactive (-IR) nerve fibers were observed in the subgemmal region and some penetrated into the taste buds. CB-LI was also detected in the cytoplasm of the spindle-shaped gustatory cells in the lower half of the trench epithelium, which contained numerous synaptic vesicles and bundles of intermediate filaments. These CB-IR gustatory cells made synapse-like contacts with CB-IR nerve terminals. Some CB-IR nerve terminals made contacts with the gustatory cells negative for CB-LI. At least three developmental stages were defined with regard to the developmental changes in the distribution of CB-LI: (1) Stage I (embryonic day (E) 18–postnatal day (P)5): CB-IR nerve fibers appeared in the lamina propria just beneath the newly-formed CVP at E18, but the gustatory epithelium of the CVP contained no CB-IR structures. Taste buds with taste pores appeared at P1. (2) Stage II (P5–10): thin CB-IR nerve fibers began entering the trench epithelium, but no CB-IR cells were observed. (3) Stage III (P10–adult): in addition to the intragemmal and perigemmal CB-IR nerve fibers, very few CB-IR cells appeared in the taste buds around P10, and their numbers increased progressively. The changes in the distribution of taste buds and CB-LI following glossopharyngeal nerve injury were similar to those observed during development. On post-operative day (PO) 4, the taste buds and CB-IR cells decreased markedly in number. These CB-IR cells became round in shape, and the number of CB-IR nerve fibers decreased markedly. On PO8, both taste buds and CB-IR cells disappeared completely. The regenerated taste buds were first observed on PO12, increased rapidly in number by PO20, and increased slowly thereafter. CB-IR nerve fibers accumulated at the subgemmal region and began penetrating into the trench wall epithelium around PO16. CB-IR cells appeared between PO20 and PO24, and their numbers increased progressively and reached the normal level on PO40. The topographical localizations of the taste buds and CB-IR cells during development and regeneration were comparable to those of normal animals. The delay of the time courses for appearance of CB-IR nerve fibers and CB-IR cells compared to the appearance of taste buds during development and regeneration suggests that CB in the gustatory epithelium may participate in the survival of the taste bud cells rather than in the induction of the taste buds.     

The time course of taste bud regeneration after glossopharyngeal or greater superficial petrosal nerve transection in rats

We previously have published data detailing the time course of taste bud regeneration in the anterior tongue following transection of the chorda tympani (CT) nerve in the rat. This study extends the prior work by determining the time course of taste bud regeneration in the vallate papilla, soft palate and nasoincisor ducts (NID) following transection of either the glossopharyngeal (GL) or greater superficial petrosal (GSP) nerve. Following GL transection in rats (n = 6 per time point), taste buds reappeared in the vallate papilla between 15 and 28 days after surgery, and returned to 80.3% of control levels (n = 12) of taste buds by 70 days postsurgery. The first appearance and the final percentage of the normal complement of regenerated vallate taste buds after GL transection resembled that seen previously in the anterior tongue after CT transection. However, in the latter case, regenerated taste buds reached asymptotic levels by 42 days after surgery, whereas within the time frame of the present study, a clear asymptotic return of vallate taste buds was not observed. In contrast to the posterior (and anterior) tongue, only 25% of the normal complement of palatal taste buds regenerated by 112 days and 224 days after GSP transection (n = 9). The difference in regenerative capacity might relate to the surgical approach used to transect the GSP. These experiments provide useful parametric data for investigators studying the functional consequences of gustatory nerve transection and regeneration.     

Epithelial overexpression of BDNF or NT4 disrupts targeting of taste neurons that innervate the anterior tongue

Brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4) are essential for the survival of geniculate ganglion neurons, which provide the sensory afferents for taste buds of the anterior tongue and palate. To determine how these target-derived growth factors regulate gustatory development, the taste system was examined in transgenic mice that overexpress BDNF (BDNF-OE) or NT4 (NT4-OE) in basal epithelial cells of the tongue. Overexpression of BDNF or NT4 caused a 93 and 140% increase, respectively, in the number of geniculate ganglion neurons. Surprisingly, both transgenic lines had severe reduction in fungiform papillae and taste bud number, primarily in the dorsal midregion and ventral tip of the tongue. No alterations were observed in taste buds of circumvallate or incisal papillae. Fungiform papillae were initially present on tongues of newborn BDNF-OE animals, but many were small, poorly innervated, and lost postnatally. To explain the loss of nerve innervation to fungiform papillae, the facial nerve of developing animals was labeled with the lipophilic tracer DiI. In contrast to control mice, in which taste neurons innervated only fungiform papillae, taste neurons in BDNF-OE and NT4-OE mice innervated few fungiform papillae. Instead, some fibers approached but did not penetrate the epithelium and aberrant innervation to filiform papillae was observed. In addition, some papillae that formed in transgenic mice had two taste buds (instead of one) and were frequently arranged in clusters of two or three papillae. These results indicate that target-derived BDNF and NT4 are not only survival factors for geniculate ganglion neurons, but also have important roles in regulating the development and spatial patterning of fungiform papilla and targeting of taste neurons to these sensory structures.