Fibroblast and epidermal growth factors modulate proliferation and neural cell adhesion molecule expression in epithelial cells derived from the adult mouse tongue

Lingual epithelial cells, including those of the taste buds, are regularly replaced by proliferative stem cells. We found that integrin beta(1), a keratinocyte stem cell marker, was expressed at the basal layer and taste buds of adult mouse tongue epithelium. We purified and cultured integrin beta(1)-positive cells (termed KT-1 cells), whose growth was stimulated by epidermal growth factor (EGF) and basic fibroblast growth factor (FGF-2). FGF-2 stimulation induced translocation of the FGF type I receptor (FGFR1) into nuclei, suggesting that the growth-stimulating effect of FGF-2 was mediated through FGFR1. EGF and FGF-2 also regulated cell surface expression of the neural cell adhesion molecule (N-CAM) in KT-1 cells. Anti-N-CAM antibody immunoreactivity was restricted to the gustatory epithelium and the nerves in the tongue epithelium, giving rise to the possibility that KT-1 may contain gustatory epithelial cells. KT-1 cells may thus be useful for analyzing the factors that regulate the growth and differentiation of lingual and gustatory epithelial cells in vitro.     

Computational model of response maps in the dorsal cochlear nucleus

The neurons in the mammalian (gerbil, cat) dorsal cochlear nucleus (DCN) have responses to tones and noise that have been used to classify them into unit types. These types (I–V) are based on excitatory and inhibitory responses to tones organized into plots called response maps (RMs). Type I units show purely excitatory responses, while type V units are primarily inhibited. A computational model of the neural circuitry of the mammalian DCN, based on the MacGregor neuromime, was used to investigate RMs of the principal cells (P-cells) that represent the fusiform and giant cells. In gerbils, fusiform cells have been shown to have primarily type III unit response properties; however, fusiform cells in the cat DCN are thought to have type IV unit response properties. The DCN model is based on a previous computational model of the cat (Hancock and Voigt Ann Biomed Eng 27: 73–87, 1999) and gerbil (Zheng and Voigt Ann Biomed Eng 34: 697–708, 2006) DCN. The basic model for both species is architecturally the same, and to get either type III unit RMs or type IV unit RMs, connection parameters were adjusted. Interestingly, regardless of the RM type, these units in gerbils and cats show spectral notch sensitivity and are thought to play a role in sound localization in the median plane. In this study, further parameter adjustments were made to systematically explore their effect on P-cell RMs. Significantly, type I, type III, type III-i, type IV, type IV-T and type V unit RMs can be created for the modeled P-cells. Thus major RMs observed in the cat and gerbil DCN are recreated by the model. These results suggest that RMs of individual DCN projection neurons are the result of specific assortment of excitatory and inhibitory inputs to that neuron and that subtle differences in the complement of inputs can result in different RM types. Modulation of the efficacy of certain synapses suggests that RM type may change dynamically.     

Effect of Gap Junction Blocker β-Glycyrrhetinic Acid on Taste Disk Cells in Frog

A gap junction blocker, 18β-glycyrrhetinic acid (β-GA), increased the membrane resistance of Ia, Ib and II/III cells of frog taste disk by 50, 160, and 300 MΩ, respectively, by blocking the gap junction channels and hemichannels. The amplitudes of gustatory depolarizing potentials in the disk cells for 4 basic taste stimuli were reduced to 40–60% after intravenous injection of β-GA at 1.0 mg/kg. β-GA of 1.0 mg/kg did not affect the resting potentials and the reversal potentials for tastant-induced depolarizing potentials in any taste disk cells. The percentage of cells responding to each of 4 basic taste stimuli and varying numbers of 4 taste qualities did not differ between control and β-GA-treated taste disk cells. This implies that gustatory depolarizing response profiles for 4 basic taste stimuli were very similar in control and β-GA-treated taste disk cells. It is concluded that β-GA at 1.0 mg/kg reduced the amplitude of gustatory depolarizing potentials in taste disk cells by strongly blocking depolarizing currents flowing through the gap junction channels and hemichannels, but probably weakly affected the gustatory transduction mechanisms for 4 taste stimuli.     

A Calcium-Receptor Agonist Induces Gustatory Neural Responses in Bullfrogs

The effect of calcium-sensing receptor (CaR) agonists on frog gustatory responses was studied using glossopharyngeal nerve recording and whole-cell patch-clamp recording of isolated taste disc cells. Calcimimetic NPS R-467 dissolved in normal saline solution elicited a large transient response in the nerve. The less active enantiomer of the compound, NPS S-467 induced only a small neural response. The EC₅₀ for NPS R-467 was about 20 μM. Cross-adaptation experiments were performed to examine the effect of 30 μM NPS R-467 and 100 μM quinine on the gustatory neural response. The magnitude of the R-467-induced response after adaptation to quinine was approximately equal to that after adaptation to normal saline solution, indicating that the receptor site for NPS R-467 is different from the site for quinine. NPS R-467 (100 μM) also induced an inward current accompanied with conductance increase and large depolarization in two (13%) of 15 rod cells, and a sustained decrease in outward current and small depolarization in six (40%) other rod cells. NPS S-467 (100 μM) induced a sustained decrease in outward current and depolarization in five (50%) of 10 rod cells. Another calcimimetic cinacalcet (100 μM) induced an inward current accompanied with conductance increase in three (27%) of 11 rod cells. The results suggest that NPS R-467 induces neural responses through cell responses unrelated to a resting K⁺ conductance decrease.     

Building sensory receptors on the tongue

Neurotrophins, neurotrophin receptors and sensory neurons are required for the development of lingual sense organs. For example, neurotrophin 3 sustains lingual somatosensory neurons. In the traditional view, sensory axons will terminate where neurotrophin expression is most pronounced. Yet, lingual somatosensory axons characteristically terminate in each filiform papilla and in each somatosensory prominence within a cluster of cells expressing the p75 neurotrophin receptor (p75NTR), rather than terminating among the adjacent cells that secrete neurotrophin 3. The p75NTR on special specialized clusters of epithelial cells may promote axonal arborization in vivo since its over-expression by fibroblasts enhances neurite outgrowth from overlying somatosensory neurons in vitro. Two classical observations have implicated gustatory neurons in the development and maintenance of mammalian taste buds—the early arrival times of embryonic innervation and the loss of taste buds after their denervation in adults. In the modern era more than a dozen experimental studies have used early denervation or neurotrophin gene mutations to evaluate mammalian gustatory organ development. Necessary for taste organ development, brain-derived neurotrophic factor sustains developing gustatory neurons. The cardinal conclusion is readily summarized: taste buds in the palate and tongue are induced by innervation. Taste buds are unstable: the death and birth of taste receptor cells relentlessly remodels synaptic connections. As receptor cells turn over, the sensory code for taste quality is probably stabilized by selective synapse formation between each type of gustatory axon and its matching taste receptor cell. We anticipate important new discoveries of molecular interactions among the epithelium, the underlying mesenchyme and gustatory innervation that build the gustatory papillae, their specialized epithelial cells, and the resulting taste buds.     

Neurotrophin-4 regulates the survival of gustatory neurons earlier in development using a different mechanism than brain-derived neurotrophic factor

The number of neurons in the geniculate ganglion that are available to innervate taste buds is regulated by neurotrophin-4 (NT-4) and brain-derived neurotrophic factor (BDNF). Our goal for the current study was to examine the timing and mechanism of NT-4-mediated regulation of geniculate neuron number during development. We discovered that NT-4 mutant mice lose 33% of their geniculate neuronal cells between E10.5 and E11.5. By E11.5, geniculate axons have just reached the tongue and do not yet innervate their gustatory targets; thus, NT-4 does not function as a target-derived growth factor. At E11.5, no difference was observed in proliferating cells or the rate at which cells exit the cell cycle between NT-4 mutant and wild type ganglia. Instead, there was an increase in TUNEL-labeling, indicating an increase in cell death in Ntf4(-/-) mice compared with wild types. However, activated caspase-3, which is up-regulated in the absence of BDNF, was not increased. This finding indicates that cell death initiated by NT-4-removal occurs through a different cell death pathway than BDNF-removal. We observed no additional postnatal loss of taste buds or neurons in Ntf4(-/-) mice. Thus, during early embryonic development, NT-4 produced in the ganglion and along the projection pathway inhibits cell death through an activated caspase-3 independent mechanism. Therefore, compared to BDNF, NT-4 plays distinct roles in gustatory development; differences include timing, source of neurotrophin, and mechanism of action.     

Converting neural signals from place codes to rate codes

 The nervous system uses two basic types of formats for encoding information. The parameters of many sensory (and some premotor) signals are represented by the pattern of activity among an array of neurons each of which is optimally responsive to a different parameter value. This type of code is commonly referred to as a place code. Motor commands, in contrast, use rate coding: the desired force of a muscle is specified as a monotonic function of the aggregate rate of discharge across all of its motor neurons. Generating movements based on sensory information often requires converting signals from a place code to a rate code. In this paper I discuss three possible models for how the brain does this. Received: 24 July 2000 / Accepted in revised form: 2 February 2001     

Efferent projection from the bed nucleus of the stria terminalis suppresses activity of taste-responsive neurons in the hamster parabrachial nuclei

Although the reciprocal projections between the bed nucleus of the stria terminalis (BNST) and the gustatory parabrachial nuclei (PbN) have been demonstrated neuroanatomically, there is no direct evidence showing that the projections from the PbN to the BNST carry taste information or that descending inputs from the BNST to the PbN modulate the activity of PbN gustatory neurons. A recent electrophysiological study has demonstrated that the BNST exerts modulatory influence on taste neurons in the nucleus of the solitary tract (NST), suggesting that the BNST may also modulate the activity of taste neurons in the PbN. In the present study, we recorded from 117 taste-responsive neurons in the PbN and examined their responsiveness to electrical stimulation of the BNST bilaterally. Thirteen neurons (11.1%) were antidromically invaded from the BNST, mostly from the ipsilateral side (12 cells), indicating that a subset of taste neurons in the PbN project their axons to the BNST. The BNST stimulation induced orthodromic responses on most of the PbN neurons: 115 out of 117 (98.3%), including all BNST projection units. This descending modulation on the PbN gustatory neurons was exclusively inhibitory. We also confirmed that activation of this efferent inhibitory projection from the BNST reduces taste responses of PbN neurons in all units tested. The BNST is part of the neural circuits that involve stress-associated feeding behavior. It is also known that brain stem gustatory nuclei, including the PbN, are associated with feeding behavior. Therefore, this neural substrate may be important in the stress-elicited alteration in ingestive behavior.     

Sensitivity of basic oscillatory mechanisms for pattern generation and detection

 Intrinsic oscillators are the basic building blocks of central pattern generators, which model the neural circuits underlying pattern generation. Coupled intrinsic oscillators have been shown to synchronize their oscillatory frequencies and to maintain a characteristic pattern of phase relationships. Recently, oscillatory neurons have also been identified in sensory systems that are involved in decoding phase information. It has been hypothesized that the neural oscillators are part of neural circuits that implement phase-locked loops (PLLs), which are well-known electrical circuits for temporal decoding. Thus, there is evidence that intrinsic neural oscillators participate in both temporal pattern generation and temporal pattern decoding. The present paper investigates the dynamics underlying forced oscillators and forced PLLs, using a single framework, and compares both their stability and sensitivity characteristics. In particular, a method for assessing whether an oscillatory neuron is forced directly or indirectly, as part of a PLL, is developed and applied to published data. Received: 17 July 2000 / Accepted in revised form: 14 March 2001     

Induced in vitro differentiation of neural-like cells from human amnion-derived fibroblast-like cells

There is growing evidence that the human amnion contains various types of stem cell. As amniotic tissue is readily available, it has the potential to be an important source of material for regenerative medicine. In this study, we evaluated the potential of human amnion-derived fibroblast-like (HADFIL) cells to differentiate into neural cells. Two HADFIL cell populations, derived from two different neonates, were analyzed. The expression of neural cell-specific genes was examined before and after in vitro induction of cellular differentiation. We found that neuron specific enolase, neurofilament-medium, beta-tubulin isotype III, and glial fibrillary acidic protein (GFAP) showed significantly increased expression following the induction of differentiation. In addition, immunostaining demonstrated that neuron specific enolase, GFAP and myelin basic protein (MBP) were present in HADFIL cells following the induction of differentiation, although one of the HADFIL cell populations showed a lower expression of GFAP and MBP. These results indicate that HADFIL cell populations have the potential to differentiate into neural cells. Although further studies are necessary to determine whether such in vitro-differentiated cells can function in vivo as neural cells, these amniotic cell populations might be of value in therapeutic applications that require human neural cells.     

Different responsiveness of the chorda tympani and glossopharyngeal nerves to L-lysine in mice

Differential taste responsiveness and functional role of thetwo taste nerves, the chorda tympani (CT) and die glossopharyngeal(GL), were studied in mice by examining neural and behavioralresponses to an essential amino acid, L-lysine (Lys). Relativeresponses to Lys were larger in the GL than in the CT nerve.The neural threshold for the Lys response was about 2.5 logunits lower in the GL (about 1.0 µM) than in the CT nerve(about 300 µM). An analysis of concentration-responserelationships suggests a possibility that there are two differentreceptors (high and low affinity types) for Lys showing differentdissociation constants. The posterior tongue region possessesboth types, while the anterior region possesses only the lowaffinity type. Behavioral aversion threshold for Lys in intact mice, measuredby use of a single bottle test, was about 1.0 µM. Thisthreshold was the same as its neural threshold in the GL nerve.Animals whose bilateral GL nerves were sectioned showed a higheraversion threshold (about 300 µM) which was the same asthe neural threshold in the CT nerve. An aversion conditionedto Lys significantly generalized to L-arginine in the intactand CT-denervated mice, and L-arginine and L-histidine in theGL-denervated mice, but the generalization pattern across varioustaste stimuli including the four basic taste stimuli (NaCl,HCl, quinine HCl and sucrose) did not prominently differ amongthe intact, the GL-denervated and CT-denervated mice. These results suggest that taste sensitivity to Lys is higherin the GL than in the CT nerve, but taste quality informationfor Lys conveyed by two taste nerves is not largely different.     

BDNF-, IGF-1- and GDNF-Secreting Human Neural Progenitor Cells Rescue Amyloid β-Induced Toxicity in Cultured Rat Septal Neurons

Alzheimer’s disease (AD) is characterized by the depositions of amyloid-β (Aβ) proteins, resulting in a reduction of choline acetyltransferase (ChAT) activity of AD brain in the early stages of the disease. Several growth factors, including brain-derived neurotrophic factor (BDNF), insulin-like growth factor (IGF)-1 and glial cell-derived neurotrophic factor (GDNF) are known to protect neuronal cell death in several neurodegenerative both in vitro and in vivo models. In this study, septal neurons were prepared from septal nucleus of embryonic (day 16-17) rat brain and treated with monomeric, oligomeric or fibrillar Aβ_1-42 peptide. Oligomeric Aβ_1-42, (10 μM) was the most potent at sublethal dose. Septal neuron cultures treated with BDNF, IGF-1 or GDNF or co-cultured with genetically modified human neural progenitor cells (hNPCs) secreting these neurotrophic factors (but not allowing contact between the two cell types), were protected from oligomeric Aβ_1-42 peptide-induced cell death, and these trophic factors enhanced cholinergic functions by increasing ChAT expression level. These results indicate the potential of employing transplanted hNPCs for treatment of AD.     

Neural differentiation of human umbilical cord matrix-derived mesenchymal cells under special culture conditions

Many neural disorders are characterized by the loss of one or several types of neural cells. Human umbilical cord-derived mesenchymal cells (hUCMs) are capable of differentiating into neuron, astroglia-like and oligodendrocyte cell types. However, a reliable means of inducing the selective differentiation of hUCMs into neural cells in vitro has not yet been established. For induction of neural differentiation, hUCMs were seeded onto sterile glass slides and six various cocktails using a base medium (DMEM/LG) supplemented with 10 % FBS, retinoic acid (RA), dimethyl sulfoxide (DMSO), epidermal growth factor (EGF) and fibroblast growth factor (FGF) were used to compare their effect on neuronal, astrocyte and oligodandrocyte differentiation. The hUCMs were positive for mesenchymal markers, while they were negative for hematopoietic markers. Differentiation to adipogenic and osteogenic lineage was detected in these cells. Our data revealed that the cocktail consisting of DMEM/LG, FBS, RA, FGF, and EGF (DF/R/Fg/E group) induced hUCM cells to express the highest percentage of nestin, ß-tubulin III, neurofilament, and CNPase. The DF/Ds/Fg/E group led to the highest percentage of GFAP expression. While the expression levels of NF, GFAP, and CNPase were the lowest in the DF group. The least percentage of nestin and ß-tubulin III expression was observed in the DF/Ds group. We may conclude that FGF and EGF are important inducers for differentiation of hUCMs into neuron, astrocyte and oligodendrocyte. RA can induce hUCMs to differentiate into neuron and oligodendrocyte while for astrocyte differentiation DMSO had a pivotal role.     

Thalamic taste responses to changing stimulus concentration

In the rat brainstem, where responses are often vigorous tobasic gustatory stimuli, the code for taste intensity appearsto be a function of total neural activity. By contrast, thalamicneurons respond sluggishly to stimuli representing the fourbasic taste qualities, and total neural activity does not risesignificantly with increasing stimulus concentration. Rather,most thalamic cells (61%) show sensitivity to concentrationchanges of one or more qualities, but a distinct minority areunaffected by intensity. Further, increases in stimulus concentrationmay affect the neurons which are sensitive either directly orinversely. Thus, only a subpopulation of thalamic neurons seemsto signal stimulus intensity, doing so through both excitatoryand inhibitory responses. The role of the thalamus in processingtaste information is discussed.     

Genetic tracing of the gustatory neural pathway originating from Pkd1l3-expressing type III taste cells in circumvallate and foliate papillae

Polycystic kidney disease 1-like 3 (Pkd1l3) is expressed specifically in sour-sensing type III taste cells that have synaptic contacts with afferent nerve fibers in circumvallate (CvP) and foliate papillae (FoP) located in the posterior region of the tongue, although not in fungiform papillae (FuP) or the palate. To visualize the gustatory neural pathways that originate from type III taste cells in CvP and FoP, we established transgenic mouse lines that express the transneuronal tracer wheat germ agglutinin (WGA) under the control of the mouse Pkd1l3 gene promoter/enhancer. The WGA transgene was accurately expressed in Pkd1l3-expressing type III taste cells in CvP and FoP. Punctate WGA protein signals appeared to be detected specifically in type III taste cells but not in other types of taste cells. WGA protein was transferred primarily to a subset of neurons located in close proximity to the glossopharyngeal (GL) nerve bundles in the nodose/petrosal ganglion (NPG). WGA signals were also observed in a small population of neurons in the geniculate ganglion (GG). This result demonstrates the anatomical connection between taste receptor cells (TRCs) in the FoP and the chorda tympani (CT) nerves. WGA protein was further conveyed to neurons in a rostro-central subdivision of the nucleus of the solitary tract (NST). These findings demonstrate that the approximately 10?kb 5'-flanking region of the mouse Pkd1l3 gene functions as a type III taste cell-specific promoter/enhancer. In addition, experiments using the pkd1l3-WGA transgenic mice reveal a sour gustatory pathway that originates from TRCs in the posterior region of the tongue.     

G-protein gamma subunit 1 is required for sugar reception in Drosophila

Though G-proteins have been implicated in the primary step of taste signal transduction, no direct demonstration has been done in insects. We show here that a G-protein gamma subunit, Ggamma1, is required for the signal transduction of sugar taste reception in Drosophila. The Ggamma1 gene is expressed mainly in one of the gustatory receptor neurons. Behavioral responses of the flies to sucrose were reduced by the targeted suppression of neural functions of Ggamma1-expressing cells using neural modulator genes such as the modified Shaker K+ channel (EKO), the tetanus toxin light chain or the shibire (shi(ts1)) gene. RNA interference targeting to the Ggamma1 gene reduced the amount of Ggamma1 mRNA and suppressed electrophysiological response of the sugar receptor neuron. We also demonstrated that responses to sugars were lowered in Ggamma1 null mutant, Ggamma1(N159). These results are consistent with the hypothesis that Ggamma1 participates in the signal transduction of sugar taste reception.     

Are mechanisms of exocytosis neurotransmitter specific?

In their review, Langley and Grant (1997) investigate the question whether mechanisms of exocytosis are neurotransmitter specific. There is now much evidence that the mechanisms governing the exocytosis of the two principal storage organelles—granules (large dense core vesicles) and electron-lucent vesicles—differ. But much less is known concerning potential differences in the release mechanisms of electron-lucent vesicles that store different types of fast neurotransmitters or of granules in different types of neurons. It is an open question whether there is a unifying control mechanism for the exocytosis of, for example, a peptide-containing granule of a glutamatergic neuron, a chromaffin granule, a noradrenergic granule or a granule from a neurosecretory neuron in the pituitary. The small electron-lucent synaptic vesicles of various kind apparently share common molecular components of regulated release. They carry the calcium sensor synaptotagmin, small GTP-binding proteins of the rab3 group or the v-SNARE synaptobrevin. Nevertheless, there may be differences in the regulatory mechanisms. This concerns the type of calcium channel involved or the absence of some of the presynaptic molecules such as rab3a, synapsin I or the t-SNAREs SNAP-25 or syntaxin from distinct types of neurons or sensory cells.     

Identification of plant volatiles activating single receptor neurons in the pine weevil (Hylobius abietis)

Naturally produced plant volatiles, eliciting responses of single olfactory receptor neurons in the pine weevil, have been identified by gas chromatography linked with mass spectrometry. The receptor neurons (n = 72) were classified in 30 types, according to the compound which elicited the strongest response in each neuron, 20 of which compounds were identified. Most potent for 14 types of neurons (n = 50) were monoterpenes, including bicyclic (e.g. α-pinene, camphor and myrtenal) for 8 types (n = 32), monocyclic (limonene, carvone, α-terpinene) for 3 types (n = 12) and acyclic (e.g. β-myrcene and linalool) for 3 types (n = 6). Other compounds eliciting strongest responses of a neuron were five sesquiterpenes, including α-copaene and a farnesene-isomer, and an anethole type which has no biosynthetic relationship with terpenes. Within one type, receptor neurons with quite selective responses to the most potent compound as well as neurons with additional responses to several, structurally similar compounds were found, indicating that the neurons may have the same functional types of membrane receptors, but different sensitivities. Response spectra of neurons within the bicyclic-, mono-cyclic and acyclic types showed more overlapping than across the neuron types. Minimal overlapping response spectra was found between monoterpene and sesquiterpene neurons. The results suggest that this structure-activity relationship is significant for encoding plant odour information in the pipe weevil. Accepted: 6 January 1997