Chikungunya infection in pregnancy - reassuring maternal and perinatal outcomes: a retrospective observational study.

OBJECTIVE: To evaluate pregnancy and neonatal outcomes, disease severity, and mother-to-child transmission of pregnant women with Chikungunya infection (CHIKV). DESIGN: Retrospective observational study. SETTING: Grenada. POPULATION: Women who gave birth during a Chikungunya outbreak between January 2014 and September 2015 were eligible. METHODS: This descriptive study investigated 731 mother-infant pairs who gave birth during a CHIKV outbreak. Women and infants underwent serological testing for CHIKV by ELISA. MAIN OUTCOME MEASURES: Primary outcomes: composite pregnancy complication (abruption, vaginal bleeding, preterm labour/cervical incompetence, cesarean delivery for fetal distress/abruption/placental abnormality or delivery for fetal distress) and composite neonatal morbidity. RESULTS: Of 416 mother-infant pairs, 150 (36%) had CHIKV during pregnancy, 135 (33%) had never had CHIKV, and 131 (31%) had CHIKV outside of pregnancy. Mean duration of joint pain was shorter among women infected during pregnancy (µ = 898 days, σ = 277 days) compared with infections outside of pregnancy (µ = 1064 days, σ = 244 days) (P < 0.0001). Rates of pregnancy complications (RR = 0.76, P = 0.599), intrapartum complications (RR = 1.50, P = 0.633), and neonatal outcomes were otherwise similar. Possible mother-to-child transmission occurred in two (1.3%) mother-infant pairs and two of eight intrapartum infections (25%). CONCLUSION: CHIKV infection during pregnancy may be protective against long-term joint pain sequelae that are often associated with acute CHIKV infection. Infection during pregnancy did not appear to pose a risk for pregnancy complications or neonatal health, but maternal infection just prior to delivery might have increased risk of mother-to-child transmission of CHIKV. TWEETABLE ABSTRACT: Chikungunya infection did not increase risk of pregnancy complications or adverse neonatal outcomes, unless infection was just prior to delivery.

Distinctive spatiotemporal expression patterns for neurotrophins develop in gustatory papillae and lingual tissues in embryonic tongue organ cultures.

Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) mRNAs are expressed in the developing rat tongue and taste organs in specific spatiotemporal patterns. BDNF mRNA is present in the early lingual gustatory papilla epithelium, from which taste buds eventually arise, prior to the arrival of gustatory nerve fibers at the epithelium, whereas NT-3 initially distributes in the mesenchyme. However, a direct test for neural dependence of neurotrophin expression on the presence of innervation in tongue has not been made, nor is it known whether the patterns of neurotrophin expression can be replicated in an in vitro system. Therefore, we used a tongue organ culture model that supports taste papilla formation while eliminating the influence from sensory nerve fibers, to study neurotrophin mRNAs in lingual tissues. Rat tongue cultures were begun at embryonic day 13 or 14 (E13, E14), and BDNF, NT-3, nerve growth factor (NGF) and neurotrophin-4 (NT-4) mRNAs were studied at 0, 2, 3 and 6 days in culture. BDNF transcripts were localized in the gustatory epithelium of both developing fungiform and circumvallate papillae after 2 or 3 days in culture, and NT-3 transcripts were in the subepithelial mesenchyme. The neurotrophin distributions were comparable to those in vivo at E13-E16. In 6-day tongue cultures, however, BDNF transcripts in anterior tongue were not restricted to fungiform papillae but were more widespread in the lingual epithelium, while the circumvallate trench epithelium exhibited restricted BDNF labeling. The NT-3 expression pattern shifted in 6-day organ cultures in a manner comparable to that in the embryo in vivo, and was expressed in the lingual epithelium as well as mesenchyme. NGF mRNA expression was subepithelial throughout 6 days in cultures. NT-4 mRNA was not detected. The neurotrophin mRNA distributions demonstrate that temporospatial localization of neurotrophins observed during development in vivo is retained in the embryonic tongue organ culture system. Furthermore, initial neurotrophin expression in the developing lingual epithelium, mesenchyme, and/or taste papillae is not dependent on intact sensory innervation. We suggest that patterns of lingual neurotrophin mRNA expression are controlled by the influence of local tissue interactions within the tongue at early developmental stages. However, the eventual loss of restricted BDNF mRNA localization from fungiform papillae in anterior tongue suggests that sensory innervation may be important for restricting the localized expression of neurotrophins at later developmental stages, and for maintaining the unique phenotypes of gustatory papillae.

Dental pulp cells produce neurotrophic factors, interact with trigeminal neurons in vitro, and rescue motoneurons after spinal cord injury.

Interactions between ingrowing nerve fibers and their target tissues form the basis for functional connectivity with the central nervous system. Studies of the developing dental pulp innervation by nerve fibers from the trigeminal ganglion is an excellent example of nerve-target tissue interactions and will allow specific questions regarding development of the dental pulp nerve system to be addressed. Dental pulp cells (DPC) produce an array of neurotrophic factors during development, suggesting that these proteins might be involved in supporting trigeminal nerve fibers that innervate the dental pulp. We have established an in vitro culture system to study the interactions between the dental pulp cells and trigeminal neurons. We show that dental pulp cells produce several neurotrophic factors in culture. When DPC are cocultured with trigeminal neurons, they promote survival and a specific and elaborate neurite outgrowth pattern from trigeminal neurons, whereas skin fibroblasts do not provide a similar support. In addition, we show that dental pulp tissue becomes innervated when transplanted ectopically into the anterior chamber of the eye in rats, and upregulates the catecholaminergic nerve fiber density of the irises. Interestingly, grafting the dental pulp tissue into hemisected spinal cord increases the number of surviving motoneurons, indicating a functional bioactivity of the dental pulp-derived neurotrophic factors in vivo by rescuing motoneurons. Based on these findings, we propose that dental pulp-derived neurotrophic factors play an important role in orchestrating the dental pulp innervation.

Molecular signaling and pulpal nerve development.

The purpose of this review is to discuss molecular factors influencing nerve growth to teeth. The establishment of a sensory pulpal innervation occurs concurrently with tooth development. Epithelial/mesenchymal interactions initiate the tooth primordium and change it into a complex organ. The initial events seem to be controlled by the epithelium, and subsequently, the mesenchyme acquires odontogenic properties. As yet, no single initiating epithelial or mesenchymal factor has been identified. Axons reach the jaws before tooth formation and form terminals near odontogenic sites. In some species, local axons have an initiating function in odontogenesis, but it is not known if this is also the case with mammals. In diphyodont mammals, the primary dentition is replaced by a permanent dentition, which involves a profound remodeling of terminal pulpal axons. The molecular signals underlying this remodeling remain unknown. Due to the senescent deterioration of the dentition, the target area of tooth nerves shrinks with age, and these nerves show marked pathological-like changes. Nerve growth factor and possibly also brain-derived neurotrophic factor seem to be important in the formation of a sensory pulpal innervation. Neurotrophin-3 and -4/5 are probably not involved. In addition, glial cell line-derived neurotrophic factor, but not neurturin, seems to be involved in the control of pulpal axon growth. A variety of other growth factors may also influence developing tooth nerves. Many major extracellular matrix molecules, which can influence growing axons, are present in developing teeth. It is likely that these molecules influence the growing pulpal axons.

Mammalian GFRalpha -4, a divergent member of the GFRalpha family of coreceptors for glial cell line-derived neurotrophic factor family ligands, is a receptor for the neurotrophic factor persephin.

Four members of the glial cell line-derived neurotrophic factor family have been identified (GDNF, neurturin, persephin, and enovin/artemin). They bind to a specific membrane-anchored GDNF family receptor as follows: GFRalpha-1 for GDNF, GFRalpha-2 for neurturin, GFRalpha-3 for enovin/artemin, and (chicken) GFRalpha-4 for persephin. Subsequent signaling occurs through activation of a common transmembrane tyrosine kinase, cRET. GFRalpha-4, the coreceptor for persephin, was previously identified in chicken only. We describe the cloning and characterization of a mammalian persephin receptor GFRalpha-4. The novel GFRalpha receptor is substantially different in sequence from all known GFRalphas, including chicken GFRalpha-4, and lacks the first cysteine-rich domain present in all previously characterized GFRalphas. At least two different GFRalpha-4 splice variants exist in rat tissues, differing at their respective COOH termini. GFRalpha-4 mRNA is expressed at low levels in different brain areas in the adult as well as in some peripheral tissues including testis and heart. Recombinant rat GFRalpha-4 variants were expressed in mammalian cells and shown to be at least partially secreted from the cells. Persephin binds specifically and with high affinity (K(D) = 6 nm) to the rat GFRalpha-4 receptor, but no cRET activation could be demonstrated. Although the newly characterized mammalian GFRalpha-4 receptor is structurally divergent from previously characterized GFRalpha family members, we suggest that it is a mammalian orthologue of the chicken persephin receptor. This discovery will allow a more detailed investigation of the biological targets of persephin action and its potential involvement in diseases of the nervous system.

Expression of sodium channel SNS/PN3 and ankyrin(G) mRNAs in the trigeminal ganglion after inferior alveolar nerve injury in the rat.

The inferior alveolar nerve is a sensory branch of the trigeminal nerve that is frequently damaged, and such nerve injuries can give rise to persistent paraesthesia and dysaesthesia. The mechanisms behind neuropathic pain following nerve injury is poorly understood. However, remodeling of voltage-gated sodium channels in the neuronal membrane has been proposed as one possible mechanism behind injury-induced ectopic hyperexcitability. The TTX-resistant sodium channel SNS/PN3 has been implicated in the development of neuropathic pain after spinal nerve injury. We here study the effect of chronic axotomy of the inferior alveolar nerve on the expression of SNS/PN3 mRNA in trigeminal sensory neurons. The organization of sodium channels in the neuronal membrane is maintained by binding to ankyrin, which help link the sodium channel to the membrane skeleton. Ankyrin(G), which colocalizes with sodium channels in the initial segments and nodes of Ranvier, and is necessary for normal neuronal sodium channel function, could be essential in the reorganization of the axonal membrane after nerve injury. For this reason, we here study the expression of ankyrin(G) in the trigeminal ganglion and the localization of ankyrin(G) protein in the inferior alveolar nerve after injury. We show that SNS/PN3 mRNA is down-regulated in small-sized trigeminal ganglion neurons following inferior alveolar nerve injury but that, in contrast to the persistent loss of SNS/PN3 mRNA seen in dorsal root ganglion neurons following sciatic nerve injury, the levels of SNS/PN3 mRNA appear to normalize within a few weeks. We further show that the expression of ankyrin(G) mRNA also is downregulated after nerve lesion and that these changes persist for at least 13 weeks. This decrease in the ankyrin(G) mRNA expression could play a role in the reorganization of sodium channels within the damaged nerve. The changes in the levels of SNS/PN3 mRNA in the trigeminal ganglion, which follow the time course for hyperexcitability of trigeminal ganglion neurons after inferior alveolar nerve injury, may contribute to the inappropriate firing associated with sensory dysfunction in the orofacial region.

Lingual BDNF and NT-3 mRNA expression patterns and their relation to innervation in the human tongue: similarities and differences compared with rodents.

Brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3) mRNAs are expressed in developing and adult rodent tongue and are important for the proper development of lingual gustatory and somatosensory innervation in rodents. Here, we wished to determine whether the findings in rodents apply to humans. By using in situ hybridization histochemistry, distinct, specific, and in some instances overlapping patterns of BDNF and NT-3 mRNA expression were found in the developing and adult human tongue, gustatory papillae, and taste buds. BDNF mRNA was expressed in the superior surface epithelium of the developing fungiform papillae (i.e., developing taste buds), in the epithelium covering the circumvallate papillae, and in the subepithelial mesenchyme. Interestingly, BDNF mRNA was expressed in the lingual epithelium before nerve fibers reached the epithelium, indicating a prespecialization of the gustatory epithelium before the arrival of nerves. In the adult fungiform papillae, BDNF mRNA labeling was found in taste buds and in restricted areas in the non-gustatory lingual epithelium. NT-3 mRNA was found in the developing lingual epithelium and gustatory papillae. NT-3 mRNA labeling was observed in the adult fungiform taste buds, overlapping with BDNF mRNA labeling, in contrast to what was seen in rodents. NT-3 mRNA was additionally found in restricted areas in filiform papillae. Protein gene product 9.5 (PGP) antibodies were used to investigate a possible correlation between lingual innervation and sites of neurotrophin gene activity. Adult human tongue innervation differed from that of rodents, possibly in part due to a different neurotrophin expression pattern in the human tongue. Based on these findings, we suggest that BDNF and NT-3 are important for the initiation and maintenance of the gustatory and somatosensory innervation also in humans. The broader and somewhat overlapping expression patterns of BDNF and NT-3 mRNAs, compared with rodents, suggest additional and possibly somewhat overlapping roles for BDNF and NT-3 in the human tongue and also indicate differences between species. It is important that interspecies differences be taken into consideration.

Glial cell line-derived neurotrophic factor receptor alpha1 availability regulates glial cell line-derived neurotrophic factor signaling: evidence from mice carrying one or two mutated alleles.

Glial cell line-derived neurotrophic factor receptor alpha1 (GFRalpha1, also known as GDNFR-alpha) is a glycolipid-anchored membrane protein of the GFRalpha family, which binds glial cell line-derived neurotrophic factor [Jing S. et al. (1996) Cell 85, 1113-1124; Treanor J. J. et al. (1996) Nature 382, 80-83], a survival factor for several populations of central and peripheral neurons, including midbrain dopamine neurons [Lin L. F. et al. (1993) Science 260, 1130-1132], and mediates its ligand-induced cell response via a tyrosine kinase receptor called Ret [Takahashi M. et al. (1988) Oncogene 3, 571-578; Takahashi M. and Cooper G. M. (1987) Molec. Cell Biol. 7, 1378-1385]. In this paper, we show that mice with a null mutation of the GFRalpha1 gene manifest epithelial-mesenchymal interaction deficits in kidney and severe disturbances of intestinal tract development similar to those seen with glial cell line-derived neurotrophic factor or Ret null mutations. There is a marked renal dysgenesis or agenesis and the intrinsic enteric nervous system fails completely to develop. We also show that newborn GFRalpha1-deficient mice display no or minimal changes in dorsal root and sympathetic ganglia. This is in contrast to the deficits reported in these neuronal populations in glial cell line-derived neurotrophic factor and Ret null mutations. Mesencephalic dopaminergic neurons in the substantia nigra and ventral tegmental area appear intact at the time of birth of the mutated mice. Mice homozygous for the GFRalpha1 null mutation die within 24 h of birth because of uremia. Heterozygous animals, however, live to adulthood. There is a significantly reduced neuroprotective effect of glial cell line-derived neurotrophic factor in such heterozygous animals, compared with wild-type littermates, after cerebral ischemia. Taken together with previous data on glial cell line-derived neurotrophic factor and Ret, our results strongly suggest that GFRalpha1 is the essential GFRalpha receptor for signaling in the glial cell line-derived neurotrophic factor-Ret pathway in the kidney and enteric nervous system development, and that GFRalpha2 or GFRalpha3 cannot substitute for the absence of GFRalpha1. Moreover, neuroprotective actions of exogenous glial cell line-derived neurotrophic factor also require full GFRalpha1 receptor expression.

Role of brain-derived neurotrophic factor in target invasion in the gustatory system.

Brain-derived neurotrophic factor (BDNF) is a survival factor for different classes of neurons, including gustatory neurons. We have studied innervation and development of the gustatory system in transgenic mice overexpressing BDNF under the control of regulatory sequences from the nestin gene, an intermediate filament gene expressed in precursor cells of the developing nervous system and muscle. In transgenic mice, the number and size of gustatory papillae were decreased, circumvallate papillae had a deranged morphology, and there was also a severe loss of lingual taste buds. Paradoxically, similar deficits have been found in BDNF knock-out mice, which lack gustatory neurons. However, the number of neurons in gustatory ganglia was increased in BDNF-overproducing mice. Although gustatory fibers reached the tongue in normal numbers, the amount and density of nerve fibers in gustatory papillae were reduced in transgenic mice compared with wild-type littermates. Gustatory fibers appeared stalled at the base of the tongue, a site of ectopic BDNF expression, where they formed abnormal branches and sprouts. Interestingly, palatal taste buds, which are innervated by gustatory neurons whose afferents do not traverse sites of ectopic BDNF expression, appeared unaffected. We suggest that lingual gustatory deficits in BDNF overexpressing mice are a consequence of the failure of their BDNF-dependent afferents to reach their targets because of the effects of ectopically expressed BDNF on fiber growth. Our findings suggest that mammalian taste buds and gustatory papillae require proper BDNF-dependent gustatory innervation for development and that the correct spatial expression of BDNF in the tongue epithelium is crucial for appropriate target invasion and innervation.

Changes in neurotrophin-3 messenger RNA expression patterns in the prenatal rat tongue suggest guidance of developing somatosensory nerves to their final targets.

While brain-derived neurotrophic factor (BDNF) messenger RNA (mRNA) has been localized in the developing gustatory epithelium, little information is available about neurotrophin-3 (NT-3) mRNA expression pattern in the prenatal developing gustatory and lingual epithelium. In the present study, using in situ hybridization histochemistry, we report on NT-3 mRNA expression in the tongue of rats. At embryonic day (E) 13-17, NT-3 mRNA was expressed subepithelially in the periphery of the developing tongue, as well as among developing muscle. At E19, there was a shift in the expression of NT-3 mRNA. It was then expressed in the surface epithelium of the developing tongue in the developing filiform papillae and, in higher concentrations, in top-surface and fringe epithelium of the developing circumvallate papillae, and top- and lateral-surface epithelium of the developing fungiform papillae. NT-3 mRNA expression in areas rich in somatosensory innervation of the tongue, as well as its specific expression in defined regions compared with BDNF, and the decreased labeling noted from prenatal and early postnatal animals to adults indicate a specific role for NT-3 in the development of lingual somatosensory innervation, as well as for maintenance of this innervation.

NGF, BDNF, NT3, NT4 and GDNF in tooth development.

Neurotrophic factors have robust effects on development, differentiation, maintenance and regeneration of neurons. In the present study, we have used in situ hybridization to determine the specific sites of gene activity of five neurotrophic factors during tooth development. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) mRNAs were mainly detected in the dental papilla/pulp in postnatal rats, and the pattern of expression correlated with onset of dental innervation. In contrast, neurotrophin 3 (NT3) and 4 (NT4) mRNA expression patterns were predominantly epithelial and were strongest during early developmental stages when teeth are not yet innervated. Glial cell line-derived neurotrophic factor (GDNF) mRNA was present in dental epithelium at early stages, but later in development, GDNF mRNA expression was mainly mesenchymal and observed in the odontoblast layer and extending into the subodontoblast zone. Our results suggest that both neurotrophins and GDNF may have multiple functions during tooth development. In addition to an influence on the establishment of the dental innervation, neurotrophic substances might have morphogenetic effects such as modulating the proliferation or differentiation of developing epithelial and mesenchymal cells.

Reparative hard tissue formation following calcium hydroxide application after partial pulpotomy in cariously exposed pulps of permanent teeth.

In a prospective study, partial pulpotomy was performed on six permanent molars with deep carious lesions and pulpal involvement. The bleeding pulp was irrigated with normal tap water until bleeding had stopped and the exposed pulp was covered with calcium hydroxide followed by zinc oxide eugenol, and finally covered with a semipermanent restoration. All teeth showed hard tissue barrier formation, both clinically and radiographically, within three months and were free from subjective and objective symptoms through the observation period (average observation period was 26 months). The patients also experienced the therapy positively. These findings and those of others have helped gain more recognition for partial pulpotomy as a strong possible alternative therapy when pulps are exposed by deep carious lesions and a bleeding pulp is exposed during the excavation process. The rationale for this therapy is to remove the infected and/or inflamed pulpal areas beneath the carious lesion and disintegrated tissue. A rapid and simplified procedure would allow the general practitioner to perform this procedure when necessary at dental clinics, without specialist facilities under conditions that avoid unnecessary contamination of the pulp.

Neurotrophic factors in the tongue: expression patterns, biological activity, relation to innervation and studies of neurotrophin knockout mice.

How taste buds develop and how they become innervated has been a matter of debate for a long time. Brain-derived neurotropic factor (BDNF) and neurotrophin-3 (NT3) mRNA expression patterns suggested a possible involvement in lingual gustatory and somatosensory innervation. Studies of null-mutated mice showed that BDNF-/- mice had few abnormal taste buds and were unable to discriminate between primary tastes. NT3-/- mice had a severe loss of lingual somatosensory innervation. These novel findings may have clinical implications in rare human conditions such as familial dysautonomia and/or in more common cases of problems with loss of taste and sensation in the mouth such as those seen after injury to the nerves, either by accident or following oral/facial surgery. Knowledge about which proteins that are required to stimulate nerve fibers to grow into mucous membranes of the oral cavity during development suggests that these same proteins might become helpful in stimulating regeneration of injured nerves in patients, perhaps helping them to regain lost taste and sensory functions. Here, the presence of glial cell-derived neurotrophic factor (GDNF) families of neurotrophic factors and receptors in the tongue is also discussed. Further, a model for the development and innervation of taste buds in mammals is proposed.

Neurturin and glial cell line-derived neurotrophic factor receptor-beta (GDNFR-beta), novel proteins related to GDNF and GDNFR-alpha with specific cellular patterns of expression suggesting roles in the developing and adult nervous system and in peripheral organs.

Cloning strategies were used to identify a gene termed glial cell line-derived neurotrophic factor receptor-beta (GDNFR-beta) related to GDNFR-alpha. In situ hybridization was then used to map cellular expression of the GDNF-related trophic factor neurturin (NTN) and GDNFR-beta mRNA in developing and adult mice, and comparisons with GDNFR-alpha and RET were made. Neurturin is expressed in postnatal cerebral cortex, striatum, several brainstem areas, and the pineal gland. GDNFR-beta mRNA was more widely expressed in the developing and adult CNS, including cerebral cortex, cerebellum, thalamus, zona incerta, hypothalamus, brainstem, and spinal cord, and in subpopulations of sensory neurons and developing peripheral nerves. NTN colocalized with RET and GDNFR-alpha in ureteric buds of the developing kidney. The circular muscle layer of the developing intestines, smooth muscle of the urether, and developing bronchiolae also expressed NTN. GDNFR-beta was found in myenteric but not submucosal intestinal plexuses. In developing salivary glands NTN had an epithelial expression, whereas GDNFR-beta was expressed in surrounding tissue. Neurturin and GDNFR-beta were present in developing sensory organs. In the gonads, NTN appeared to be expressed in Sertoli cells and in the epithelium of the oviduct, whereas GDNFR-beta was expressed by the germ cell line. Our findings suggest multiple roles for NTN and GDNFR-beta in the developing and adult organism. Although NTN and GDNFR-beta expression patterns are sometimes complementary, this is not always the case, suggesting multiple modi operandi of GDNF and NTN in relation to RET and the two binding proteins, GDNFR-alpha and GDNFR-beta.

Cellular and developmental patterns of expression of Ret and glial cell line-derived neurotrophic factor receptor alpha mRNAs.

Glial cell line-derived neurotrophic factor (GDNF) has recently been shown to signal by binding to GDNF receptor-alpha (GDNFR-alpha), after which the GDNF-GDNFR-alpha associates with and activates the tyrosine kinase receptor Ret. We have localized Ret messenger RNA (mRNA) in the developing and adult rodent and compared with to the expression of GDNF and GDNFR-alpha mRNA. Ret mRNA is strongly expressed in dopamine neurons and alpha-motor neurons as well as in thalamus, ruber and occluomotor nuclei, the habenular complex, septum, cerebellum, and brain stem nuclei. Ret mRNA was also found in several sensory systems, in ganglia, and in nonneuronal tissues such as teeth and vibrissae. Very strong Ret mRNA signals are present in kidney and the gastrointestinal tract, where Ret and GDNF mRNA expression patterns are precisely complementary. The presence of Ret protein was confirmed in adult dopamine neurons using immunohistochemistry. GDNFR-alpha mRNA was strongly expressed in the developing and adult dopamine neurons. It was also found in neurons in deep layers of cortex cerebri, in hippocampus, septum, the dentate gyrus, tectum, and the developing spinal cord. In the kidney and the gastrointestinal tract, GDNFR-alpha mRNA and Ret mRNA distribution overlapped. Dorsal root ganglia, cranial ganglia, and developing peripheral nerves were also positive. GDNFR-alpha was additionally found in sensory areas and in developing teeth. Sensory areas included inner ear, eye, olfactory epithelium, and the vomeronasal organ, as well as developing tongue papillae. The temporospatial pattern of expression of GDNFR-alpha mRNA did not always match that of Ret mRNA. For instance, GDNFR-alpha mRNA was also found in the developing ventral striatum, including the olfactory tubercle, and in hippocampus. These areas seemed devoid of Ret mRNA, suggesting that GDNFR-alpha might also have functions unrelated to Ret.

Lingual deficits in BDNF and NT3 mutant mice leading to gustatory and somatosensory disturbances, respectively.

A combination of anatomical, histological and physiological data from wild-type and null-mutated mice have established crucial roles for BDNF and NT3 in gustatory and somatosensory innervation of the tongue, and indeed for proper development of the papillary surface of the tongue. BDNF is expressed in taste buds, NT3 in many surrounding epithelial structures. Absence of BDNF in mice leads to severely malformed taste bud-bearing papillae and severe reduction of taste buds, a loss of proper innervation of remaining taste buds and a loss of taste discrimination although not of the suckling reflex per se. In contrast, absence of NT3 leads to a massive loss of somatosensory innervation of lingual structures. These findings demonstrate distinct roles for BDNF and NT3 in the establishment of the complex innervation apparatus of the tongue with non-overlapping roles for the lingual gustatory and somatosensory systems. The distinction between different sensory modalities, being dependent on either BDNF or NT3 may also have clinical implications.

Cellular expression of neurotrophin mRNAs during tooth development.

Target-derived neurotrophins support and sustain peripheral sensory neurons during development. In addition, it has been suggested that these growth factors could have developmental functions in non-neuronal tissues. To further elucidate the possible roles of neurotrophins in tooth morphogenesis and innervation, we have used in-situ hybridization to determine the specific sites of neurotrophin gene activity in pre- and postnatal rat jaws from E16 to P7. All four neurotrophins were expressed during tooth development with specific temporospatial patterns. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) mRNAs were mainly detected in the dental papilla/pulp in postnatal animals, and the pattern of expression correlated with the onset of dental innervation. In contrast, neurotrophin 3 (NT3) and neurotrophin 4 (NT4) mRNA expression patterns were predominantly epithelial and were strongest during early developmental stages when teeth are not yet innervated. Dental papilla NGF-mRNA expression was first seen in both epithelium and mesenchyme and later shifted to the odontoblast layer and the subodontoblast zone. BDNF-mRNA labeling was present in low levels in the early dental organ, but increased in the pulp and in the odontoblast cell layer of the developing teeth at later developmental stages. Both NT3 and NT4 mRNA were observed in the prenatal oral epithelium and the inner dental epithelium. NT3-mRNA labeling was seen mainly in the cervical loop region, fissure system depressions and cuspal tops, while NT4 mRNA was more evenly distributed in the dental epithelium. At P7, NT3-mRNA labeling was below detection level and NT4 mRNA expression was lower than at prior stages. Complementary to reports on the presence of low-affinity neurotrophin receptor (LANR), trkB and trkC mRNA in the developing teeth, our results suggest that neurotrophins may have multiple functions during tooth morphogenesis. Neurotrophins might participate in epithelial-mesenchymal interactions in early tooth morphogenetic events such as proliferation and differentiation of epithelial and mesenchymal cells. In addition, based on mRNA localization in postnatal animals, we also suggest that NGF and BDNF (beside glial cell line-derived neurotrophic factor) might participate in establishing and maintaining the innervation of the teeth, thus acting as classical neurotrophic factors.

Differential expression of brain-derived neurotrophic factor and neurotrophin 3 mRNA in lingual papillae and taste buds indicates roles in gustatory and somatosensory innervation.

Although many studies have demonstrated the dependency of taste bud function and/or survival on intact innervation, relatively few have dealt with the development of taste bud innervation. Using in situ hybridization histochemistry, we show that brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) mRNA are expressed in a specific pattern in the taste buds, tongue papillae, and lingual epithelium during development and that expression persists into adulthood. BDNF mRNA is expressed in a fraction of the taste cells of the developing and adult taste buds in rats, showing different labeling intensities among the labeled cells. NT3 and mRNA seems to be located in areas other than those where BDNF mRNA is expressed, mainly in the superior epithelial surfaces of circumvallate papillae, the outer surface epithelium of foliate papilae, the superior surface and the lateral epithelium of the fungiform papillae, and the epithelium of the filiform papillae. NT3 mRNA labeling is also observed among muscle and connective tissue of the tongue. The morphological appearance, expression of NT3 mRNA, and ramification of nerve fibers in defined epithelial structures in the posterior wall of the anterior filiform papillae suggest the existence of a mechanosensory apparatus in these papillae. Nerve growth factor and neurotrophin 4 probes did not give rise to selective labeling in tongue, although their presence cannot be totally excluded. Based on present and prior studies, we suggest that BDNF is needed during initiation and for maintenance of gustatory innervation of taste buds and gustatory papillae and that NT3 is mainly needed for somatosensory innervation of the tongue.

Cellular expression of GDNF mRNA suggests multiple functions inside and outside the nervous system.

Glial-cell-line-derived neurotrophic factor (GDNF) is a distant member of the transforming growth factor-beta family and has potent neurotrophic effects on several classes of neurons including dopamine neurons and motoneurons. Here, we have used in situ hybridization to describe the development of the cellular expression of GDNF mRNA pre- and postnatally. Consistent with dopaminotrophic activity, GDNF mRNA is expressed in the developing basal ganglia and the olfactory tubercle. It is also found in a thalamic nucleus, in neurons of the substantia innominata, in the developing Purkinje neurons and the developing locus coeruleus area, and in trigeminal brainstem nuclei. In the spinal cord, neuronal expression is found in Clarke's column. GDNF mRNA is also expressed in the dorsal horns during development. Additional GDNF mRNA expression in the head region includes the carotid body, the retina, the vibrissae, the inner ear, the ear canal, and epithelium in the nasal cavity. Prominent expression is also found in the developing teeth. The widespread expression of GDNF in developing skeletal muscle is consistent with trophic activity on alpha-motoneurons. The smooth muscle layers of the gastrointestinal tract are also strongly positive. A very strong signal is found in the outer mesenchyme of the developing metanephric kidney. We conclude that GDNF mRNA is expressed in many different cellular systems inside and outside the central nervous system during development, suggesting multiple functions of GDNF in the developing organism.

Brain-derived neurotrophic factor mRNA is expressed in the developing taste bud-bearing tongue papillae of rat.

Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are expressed in many areas of the nervous system and its target tissues. Using in situ hybridization we have investigated the possible presence of NGF mRNA and BDNF mRNA in the developing fungiform and circumvallate papillae of the rat tongue. BDNF mRNA is present in the epithelium of the developing fungiform papillae in E15, E16, and E17 rat embryos with peak concentration at E16. It starts to diminish after E17 and is almost absent at E21. There is a specific temporospatial change in the expression of BDNF mRNA in developing circumvallate papillae. It is expressed in the epithelium of the superior and posterior surfaces of the papillae at E15, E16, and E17. Already at E17 the BDNF mRNA labeling has started to decrease in the superior epithelium. At E19 and E21, BDNF mRNA is exclusively present in the epithelium of the inner and outer walls of the trench, surrounding the papilla at the posterior and lateral surfaces where the taste buds are located later in life. BDNF mRNA was also detected in the developing palatal taste buds. NGF mRNA was below detection level in the developing papillae. The highly localized expression of BDNF mRNA in areas where taste buds are to be formed suggests that BDNF may be one crucial factor in the formation of the epithelial innervation prior to taste bud formation. It might also participate in the formation and/or maintenance of the papillary and/or taste bud innervation apparatus. We conclude that the neurotrophin BDNF is expressed in early development of taste bud-bearing papillae in the rat tongue in a temporally and spatially controlled manner, presumably to act as a target-derived chemoattractant for the early nerve fibers.