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J Represa

Publications and source records attributed to J Represa.

At least 37 records · Page 2Linked to original sources
PubMed1995

Insulin-like growth factor-I regulates cell proliferation in the developing inner ear, activating glycosyl-phosphatidylinositol hydrolysis and Fos expression.

The role of insulin-like growth factors (IGF) was investigated during the early development of the inner ear. IGF-I stimulated growth of otic vesicles that were isolated and cultured in vitro. IGF-I induced DNA synthesis, increased cell number, and mitotic rate in a dose-dependent manner at concentrations between 0.1-10 nM. IGF-II also induced growth but with a lower potency, whereas insulin had no effect. In the presence of IGF-I, otic vesicles developed from stage 18 to stage 21 in 24-h cultures, mimicking the normal mitotic pattern and morphogenesis in vivo. IGF-I also stimulated growth in the cochleovestibular ganglion. Binding of 125I-IGF-I to specific receptors occurred with high affinity. An autoradiographic study of sections from otic vesicles showed radiolabeled IGF-I in the epithelium. Immunoreactivity to IGF-I was detected in the otic vesicle and in the cochleovestibular ganglion. Intracellular signaling mechanisms of IGF were explored by studying the turnover of glycosylated phosphatidylinositols and the expression of Fos oncoprotein. IGF-I rapidly increased Fos levels in cultured otic vesicles. Furthermore, antisense oligonucleotides complementary to c-fos were able to inhibit IGF-I-induced growth. Both IGF-I-induced cell proliferation and Fos expression were blocked by an antiinositol phosphoglycan (alpha-IPG) antibody. This work suggests that IGF-I may be a candidate to regulate proliferative growth of the otic primordium during normal development and that this action requires the sequential modulation of glycosyl-phosphatidylinositol turnover and Fos expression.

Animals↗
PubMed1995

Developing inner ear sensory neurons require TrkB and TrkC receptors for innervation of their peripheral targets.

The trkB and trkC genes are expressed during the formation of the vestibular and auditory system. To elucidate the function of trkB and trkC during this process, we have analysed mice carrying a germline mutation in the tyrosine kinase catalytic domain of these genes. Neuroanatomical analysis of homozygous mutant mice revealed neuronal deficiencies in the vestibular and cochlear ganglia. In trkB (-/-) animals vestibular neurons and a subset of cochlear neurons responsible for the innervation of outer hair cells were drastically reduced. The peripheral targets of the respective neurons showed severe innervation defects. A comparative analysis of ganglia from trkC (-/-) mutants revealed a moderate reduction of vestibular neurons and a specific loss of cochlear neurons innervating inner hair cells. No nerve fibres were detected in the sensory epithelium containing inner hair cells. A developmental study of trkB (-/-) and trkC (-/-) mice showed that some vestibular and cochlear fibres initially reached their peripheral targets but failed to maintain innervation and degenerated. TrkB and TrkC receptors are therefore required for the survival of specific neuronal populations and the maintenance of target innervation in the peripheral sensory system of the inner ear.

Animals↗
PubMed1995

Differential effects of combined trk receptor mutations on dorsal root ganglion and inner ear sensory neurons.

We have generated double mutant mice deficient in pairs of two different Trk receptors and have analysed the effects on survival and differentiation of dorsal root ganglion (DRG), inner ear cochlear and vestibular sensory neurons. In most combinations of mutant trk alleles, the defects observed in double compared to single mutant mice were additive. However, double homozygous trkA-/-;trkB-/- DRG and trkB-/-;trkC-/- vestibular neurons showed the same degree of survival as single trkA-/- and trkB-/- mice, respectively, suggesting that those neurons required both Trk signaling pathways for survival. In situ hybridisation analysis of DRG neurons of double mutant mice revealed differential expression of excitatory neuropeptides. Whereas calcitonin-gene-related peptide expression correlated with the trkA phenotype, substance P expression was detected in all combinations of double mutant mice. In the inner ear, TrkB- and TrkC-dependent neurons were shown to at least partially depend on each other for survival, most likely indirectly due to abnormal development of their common targets. This effect was not observed in DRGs, where neurons depending on different Trk receptors generally innervate different targets.

Animals↗
PubMed1994

Immunohistochemical localization of calcium-binding proteins in the human cutaneous sensory corpuscles.

The localization of the calcium-binding proteins (CaBP) calbindin-D28k (CB), parvalbumin (PV) and S-100 protein (S100P) in the human cutaneous sensory corpuscles was studied by immunohistochemical procedure using monoclonal antibodies. Occurrence of CB, PV and S100P immunoreactivity (IR) was observed in the lamellar cells of Meissner's corpuscles. In the pacinian corpuscles, S100P IR was restricted to the inner-core cells whereas CB and PV IR were found labelling the axon, inner core, outer core and capsule. At the light-microscope level of resolution, the presence of IR in the axon of Meissner's corpuscles cannot be ensured. Since calcium ions (Ca2+) seem to participate in the mechanoreceptor electrogenesis, present results suggest that CaBP could be involved in buffering and/or transport of Ca2+ within the specialized cells surrounding the axon tips of sensory corpuscles, thus, maintaining the periaxonal microenvironment.

Adult↗
PubMed1994

Pattern of trkB protein-like immunoreactivity in vivo and the in vitro effects of brain-derived neurotrophic factor (BDNF) on developing cochlear and vestibular neurons.

The cochleo-vestibular ganglion (CVG) contains the neurons connecting the sensory epithelia of the inner ear to the cochlear and vestibular nuclei in the medulla. Expression of trkB protein-like immunoreactivity was studied in the developing CVG, using both Western blot and immunocytochemistry on tissue sections. Specific immunoreactivity was observed in the CVG from the 12th gestation day (gd) to the first postnatal week, reflecting the presence of high-affinity receptors for brain-derived neurotrophic factor (BDNF), a member of the NGF family of neurotrophins. Whole explants and dissociated cell cultures of cochlear (CG) and vestibular ganglion (VG) from mouse embryos and postnatal specimens were grown in neurotrophin-free medium to assay changes in neurite outgrowth and neuronal survival in response to the addition of physiological concentrations (0-5 ng/ml) of BDNF. Exogenous BDNF (2 ng/ml) promoted neurite outgrowth and neuronal survival in explants of both CG and VG, and the effects were stage-dependent. The onset of the response to BDNF occurred at gd 11-12. The response then reached a maximum between 14 and 18 gd and subsequently decreased, although it remained significantly present during the first postnatal week. BDNF-induced response was no longer observed in the mature cochlear and vestibular ganglion (after 30 postnatal days). The effects of BDNF on neuronal differentiation and survival were dose-dependent, starting at 0.5 ng/ml, with saturation at 2 ng/ml and half-maximal effect occurring between 1 and 1.5 ng/ml. On the basis of our results, we propose that BDNF may be physiologically involved in the control of both neuronal differentiation, and central and peripheral target-dependent neuronal death, in the CVG of embryos and early postnatal mice. BDNF may act alone or in cooperation with other neurotrophins to establish the afferent innervation of the inner ear sensory epithelium.

Animals↗
PubMed1994

Developmental changes in nerve growth factor (NGF) binding and NGF receptor proteins trkA and p75 in the facial nerve.

This study characterizes the temporal-spatial distribution of nerve growth factor (NGF) low (p75) and high-affinity (trkA) receptors in the facial nerve and geniculate ganglion (GG) of developing quail embryos (E-3 to E-14). We used 125I-labeled NGF (125I-NGF) to study binding dynamics in a temporal series of isolated primordia and an autoradiographic series of staged specimens to characterize the occurrence and distribution of NGF receptors in this cranial nerve and its ganglion. In addition, expression of trkA and p75 protein-like immunoreactivity in the facial nerve and GG was studied by Western blot, in order to distinguish between high- and low-affinity NGF receptors respectively. The quantitative study of binding show that isolated facial primordia ranging from E-3 to E-14 exhibit different levels of specific binding. High initial binding levels were observed on E-3 specimens, then an initial decrease on day 4 (E-4) followed by a steady increase from days E-4 to E-7. Maximum 125I-NGF binding was achieved on E-7, followed by a steady decline in binding on days 8 (E-8) and 9 (E-9), reaching near background levels on day 10 (E-10) of development and until the oldest stage assayed (E-14). Most of the cells bearing NGF receptors appeared to be non-neuronal crest-derived cells, but some placode-derived neurons and motor fibers of the VIIth cranial nerve transiently expressed the ability to bind 125I-NGF. The temporal pattern of p75 expression matches the pattern of quantitative binding of NGF, while the trkA expression is restricted to a few stages mainly E7 and E9, implying that most of the binding detected is via low-affinity receptors, except for a proportion of high-affinity receptors present at stages of maximum binding. This temporal pattern of NGF binding sites suggests that cells within the VIIth cranial nerve are responsive to and/or dependent upon NGF in vivo, so NGF may play a biological role during normal development of the facial nerve. In view of the developmental events that parallel the occurrence and type of NGF binding sites, we suggest that this role may be to modulate from earlier chemotaxis and cell proliferation to much later events, such as neuronal differentiation and neuron-glia interactions. The significance of these findings in regeneration during adult life remain to be investigated.

Animals↗
PubMed1993

Brain-derived neurotrophic factor and neurotrophin-3 induce cell proliferation in the cochleovestibular ganglion through a glycosyl-phosphatidylinositol signaling system.

We have investigated the role of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in the regulation of cell proliferation in the early developing cochleovestibular ganglion (CVG). Ganglia were isolated from 72-hr chick embryos and cultured for 24 hr. Both BDNF and NT-3 had a powerful mitogenic effect, at doses of 1-5 ng/ml, consistent with an involvement of the high-affinity receptor. Evidence for the participation of the glycosyl-phosphatidylinositol (GPI)/inositol phosphoglycan (IPG) signaling system in the mediation of proliferative effects of BDNF and NT-3 is presented. Both of these neurotrophins elicited a fast and transient hydrolysis of labeled GPI, approximately 60% in 30 sec. The dose-response profile of GPI hydrolysis overlaps the neurotrophin-induced cell proliferation response profile. Anti-IPG antibodies were able to block the growth-promoting effects of BDNF and NT-3. Anti-IPG antibodies immunoprecipitated a CVG-endogenous IPG, induced upon BDNF treatment, which exhibited proliferative stimulating properties. Both BDNF and NT-3 are proposed as potential candidates for regulation of growth during CVG development, with this mitogenic effect being mediated by the GPI/IPG signaling system.

Animals↗
PubMed1993

Brain-derived neurotrophic factor and neurotrophin-3 support the survival and neuritogenesis response of developing cochleovestibular ganglion neurons.

The effects of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) on the differentiation of avian cochleovestibular ganglion and their possible association with the hydrolysis of glycosyl-phosphatidylinositol (GPI) were studied. BDNF and NT-3 (2 ng/ml) promoted neurite outgrowth in explants of both cochlear and vestibular ganglia. This effect on neuritogenesis was stage-dependent, reaching a maximum at E7 for NT-3 and at E9 for BDNF. The magnitude of the response of the vestibular ganglion to BDNF was always smaller than that of the cochlear ganglion of an equivalent stage. BDNF and NT-3 stimulation of neuronal survival and neurite extension was also demonstrated in dissociated neuronal cell cultures. The effect was concentration-dependent with saturation of the response occurring at 4 ng/ml for BDNF and at 2 ng/ml for NT-3, the half-maximal effect occurring at 2 and 1 ng/ml, respectively, for the most sensitive stages of the chick cochlear ganglion. Inositol phosphoglycan (IPG) did not mimic the effects of BDNF or NT-3 on neuronal survival and neurite outgrowth, nor was it able to potentiate their responses. Antibodies raised against IPG did not block the effects of these neurotrophins. The results suggest that BDNF and NT-3 may act in cooperation to establish the innervation pattern of the inner ear. Unlike their early proliferative effects, neurotrophic effects are uncoupled from the GPI/IPG signal transduction system.

Animals↗
PubMed1992

Myb p75 oncoprotein is expressed in developing otic and epibranchial placodes.

The c-myb proto-oncogene encodes a transcriptional regulatory protein which is highly conserved throughout evolution. Myb has been considered to be normally restricted to hematopoietic tissues, but there are indications that this might not always be the case. The present work shows the expression of a p75 Myb oncoprotein in the otic vesicle and epibranchial placodes of the early chick embryo. Expression was sequential and followed the same time course as the formation of placode-derived cranial ganglia. The results suggest a potential role for c-myb in regulation of placode development and neurogenesis.

Animals↗
PubMed1992

Development of Na(+)- and K(+)-currents in the cochlear ganglion of the chick embryo.

The development of Na(+)- and K(+)-currents in the primary afferent neurons of the cochlear ganglion was studied using the patch-clamp technique. Cells were dissociated between days 6 and 17 of development and membrane currents recorded within the following 24 h. Outward currents were the first to appear between days 6 and 7 of embryonic development and their magnitude increased throughout development from 200 pA on day 7 to 900 pA on days 14-16. Threshold for activation decreased by 20 mV between days 8 and 14. Outward currents were absent when Cs+ replaced K+ in the pipette and were partially blocked by external tetraethylammonium. Outward currents contained at least three components: (i) a non-inactivating outward current, similar to the delayed-rectifier, predominating in mature neurons; (ii) a slowly inactivating current (tau about 200 ms), most evident in early and intermediate stages (days 7-10); and (iii) a rapidly inactivating outward current (tau about 20 ms) similar to the A-current (IA) described in other neurons, which was distinctly expressed in mature neurons. Sodium currents were identified as fast transient inward currents, sensitive to tetrodotoxin and extracellular Na(+)-removal. They appeared later than K(+)-currents and increased in size from about 100 pA between days 9-11 to 600 pA by days 13-16. The development of membrane currents in cochlear ganglion neurons corresponded to defined stages of the innervation pattern of the chick cochlea [Whitehead and Morest (1985) Neuroscience 14, 255-276]. These currents could be functionally related to the establishment of synaptic connections between transducing cells and primary afferent neurons.

Animals↗
PubMed1992

Transient outward currents in cochlear ganglion neurons of the chick embryo.

Cochlear ganglion neurons were isolated from chick embryos and membrane currents recorded using the patch-clamp technique. Depolarizing voltage steps elicited transient outward currents whose inactivation was best fitted by a double-exponential function with time constants < 30 ms and > 100 ms. The fast inactivating transient outward current (Ito,f) had a threshold for activation of -61 +/- 5.5 mV; steady-state inactivation was voltage-dependent between -90 and -60 mV, with half-inactivation near -75 mV. The slowly inactivating outward current (Ito,s) showed an activation threshold of 34 +/- 4 mV. Half-inactivation was at -67 +/- 3 mV. Ito,f was blocked by 4-aminopyridine which did not affect Ito,s. The effect was concentration- and voltage-dependent. Tetraethylammonium had no effect on either fast or slow transient currents but reduced the amplitude of the non-inactivating outward current in a dose-dependent manner. Ito,f was strongly inhibited by removing Ca2+ from the extracellular bathing solution. Cobalt ions inhibited Ito,f in a dose-dependent manner between 2 and 20 mM. The inhibitory effect of Co2+ was voltage-dependent, displaying a bell-shaped inhibition curve as a function of membrane voltage, maximal inhibition occurring between -20 and 0 mV. Ca2+ removal did not affect Ito,s and partially reduced the amplitude of the steady-state current. These results provide kinetic and pharmacological evidence for the presence of two distinct transient outward currents in cochlear neurons. These currents may play a role in the first synaptic relay of sound transmission.

4-Aminopyridine↗
PubMed1992

[Effect of hair cells and nerve growth factor on the differentiation of neurons of the cochleovestibular ganglion].

This study address the effect of hair cells, as target tissues and the nerve growth factor (NGF) on the survival and differentiation of murine cochleovestibular ganglion neurons. Isolated ganglia, ganglia with otic sensory epithelium and ganglia plus NGF were explanted from 12 gestation day mouse embryo and cultured for 14 days, in M-199 plus 10% fetal calf serum, on collage gel substrata. Histological and immunocytochemical studies, searching for cell degeneration and neurofilament (160,000 daltons) expression, have shown that there were significantly less survival as well as fewer differentiated cochleovestibular ganglion neurons in isolated ganglion explants, than either in explants of ganglia with hair cells or in those contained NGF (50 ng/ml) as a trophic factor. Our results confirm that hair cells exert a trophic effect not only on neuronal survival but on neuronal cytodifferentiation, and that the influence of hair cells on cochleovestibular ganglion neurons is mediated by nerve growth factor. A role for NGF on regeneration in the auditory system is suggested.

Animals↗
PubMed1992

[Childhood rhabdomyosarcoma of nasolabial localization. Apropos of a case].

A case of embryonal Rhabdomyosarcoma of nasolabial location--very infrequent site of appearance--in an eleven year-old-child is presented. After the AA' review of the subject, they drawn out the conclusion that systemic combined therapy--surgery, irradiation and chemotherapy--offers the best chances of survival for this condition.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1991

The int-2 proto-oncogene is responsible for induction of the inner ear.

The int-2 proto-oncogene encodes several products related to the fibroblast growth factor (FGF) family. FGFs have been associated with mesoderm induction in the amphibian embryo and int-2 has a distinct pattern of expression throughout development in vertebrates. But evidence for a function of int-2 in embryo-genesis has been lacking. In the mouse embryo, int-2 transcripts have been detected in the rhombencephalon at a developmental stage where classical experiments showed that the induction of the inner ear occurs. This raises the possibility that int-2 may constitute a signal for the induction of the otic vesicle, the primordium of the inner ear. We provide direct evidence for this view by showing that (1) the formation of the otic vesicle is inhibited by antisense oligonucleotides targeted to the secreted form of int-2, and by antibodies against int-2 oncoproteins, and (2) basic FGF (bFGF) can mimic the inductive signal in the absence of the rhombencephalon.

Animals↗
PubMed1991

Glycosyl-phosphatidylinositol/inositol phosphoglycan: a signaling system for the low-affinity nerve growth factor receptor.

Nerve growth factor (NGF) exerts a variety of actions during embryonic development. At the early stages of inner ear development, NGF stimulates cell proliferation, an effect mediated through low-affinity receptors. We have studied the possibility that the glycosyl-phosphatidylinositol/inositol phosphoglycan (glycosyl-PtdIns/IPG) system is involved in transmitting this NGF signal. Endogenous glycosyl-PtdIns was characterized in extracts of cochleovestibular ganglia (CVGs) that incorporated [3H]glucosamine, [3H]galactose, [3H]myristic acid, and [3H]palmitic acid. Incubation of CVG with NGF produced a rapid and transient hydrolysis of glycosyl-PtdIns. Hydrolysis was complete at 100 ng/ml, and the half-maximal effect occurred at 25 ng/ml, overlapping with the concentration dependence of the mitogenic effect of NGF. An IPG was isolated from embryonic extracts. It had biological effects similar to those reported for the insulin-induced IPG in other tissues. It exerted a powerful mitogenic effect on CVG, comparable to that of NGF. Both the IPG- and NGF-induced cell proliferation were blocked by anti-IPG antibodies that recognized the endogenous IPG on a silica plate immunoassay. These results show that CVG possesses a fully active glycosyl-PtdIns/IPG signal transduction system and that the proliferative effects associated with NGF binding to low-affinity receptors require IPG generation.

Animals↗
PubMed1991

Temporal pattern of nerve growth factor receptor expression in developing cochlear and vestibular ganglia in quail and mouse.

We have previously demonstrated the presence of specific receptors for nerve growth factor (NGF) in cochleovestibular ganglia of 72 h (stage 19-20) quail embryos, with a greater density of NGF receptors in the cochlear portion of the ganglion. The present study was conducted to determine the temporal pattern of NGF receptor expression in cochlear and vestibular ganglia throughout development, and was conducted in two species, quail and mouse. As in the quail, specific binding of 125I-NGF was detected in cochleovestibular ganglia of mouse embryos from an embryonic age equivalent to 72 h quail embryos (embryonic day 11, E11), with a similar concentration of 125I-NGF binding in the cochlear portion. Quantitative studies revealed that 125I-NGF binding continued to increase, in both cochlear and vestibular ganglia, for several days of development, and then began to decrease to minimal levels. Maximal levels were achieved at E7 in the quail, and E14 to E16 in the mouse, while minimal levels were reached by E13 in the quail, and E18 in the mouse. The level of 125I-NGF binding in cochlear ganglia was two to three times higher than in vestibular ganglia; a finding corroborated by radioautographic studies. In both quail and mouse, NGF receptors were more heavily concentrated in the ventromedial portion of the cochlear ganglion, adjacent to the cochlear duct; an area containing both support cells and peripheral neuronal processes. In the vestibular ganglion, 125I-NGF binding was more homogeneous, although small areas containing high densities of silver grains were observed. The presence of NGF receptors in cochlear and vestibular ganglia suggests that these ganglia may be responsive to and/or dependent upon NGF during their development.

Animals↗
PubMed1991

Inositol phospho-oligosaccharide stimulates cell proliferation in the early developing inner ear.

The ability of an inositol phospho-oligosaccharide (POS) to mimic the mitogenic effects of nerve growth factor (NGF) and insulin on the early development of the inner ear was investigated. POS (10 microM) stimulated the incorporation of [3H]thymidine into the cochleovestibular ganglion by 3.9-fold. NGF (50 ng/ml) stimulation was 4.7-fold. POS and NGF showed no additivity. Cells induced to proliferate by POS overlapped with those expressing NGF receptors. POS, like insulin, potentiated the mitogenic effect of bombesin on the otic vesicle epithelium. DNA synthesis in the presence of bombesin (100 nM) plus POS (10 microM) was increased by 6.4-fold. POS stimulation was not additive with insulin. The results suggest that POS may play a role in growth factor regulation of cell proliferation during embryonic development.

Animals↗