Potential of some autochthonous wild plants of Burundi for vegetable oil and valuable compounds production.

Twelve species of indigenous plants have been studied in order to valorize some natural resources of Burundi (Eastern Africa) to investigate possibilities of vegetable oil production. Physicochemical properties and oil contents were determined from seeds harvested through five ecogeographic zones. From oilcake extracts, total sugars contents, proteins (TPrC), polyphenolic (TPhC), and flavonoids were quantified using spectrophotometry. Furthermore, antioxidant activity of oilcake extracts was assessed by 2, 2-diphenyl-b-picrylhydrazyl (DPPH) radical-scavenging and ferric reducing antioxidant power (FRAP) assays. All oil contents obtained were found to be quite similar to those of common oleaginous seeds. The two highest were found in Parinari curatellifolia (61.44 ± 4.81% Dry Matter) and Myrianthus arboreus (48.26 ± 5.96% DM). More than half of the species have shown TPrC ranging from 10 to 24% dry matter of oilcake (DM). Brachystegia longifolia was revealed exceptionally stronger antioxidant potential: effectiveness antiradical of 163.06 ± 26.29 mL/µg.min (DPPH assay) and reducing power of 2618.21 ± 161.22 GAE/100 g DM (FRAP assay). TPhC were positively correlated (p < 0.05) to the antioxidant activity. This pioneering work on these wild species highlight the potential for producing vegetable oil and valuable biomolecule sources likely for food, cosmetics, pharmacy and industry.

[Amyloid goiter: first sign of systemic amyloidosis]. / Goitre amyloïde : première manifestation d'une amylose systémique.

Although amyloid infiltration of the thyroid gland is an uncommon but well-known phenomenon, the appearance of a goiter secondary to amyloid deposits is rare. The goiter enlarges rapidly and progressively, often becoming compressive like thyroid cancer. The diagnosis is rarely suggested clinically even in the presence of known amyloidosis. We describe the case of a 45-year-old patient who presented an amyloid goiter as the first manifestation of systemic amyloidosis, probably secondary to bronchiectasis.

[Beta interferon and thyroid]. / Interféron bêta et thyroïde.

We report the case of a thyroiditis in a 44-year-old woman with relapsing-remitting multiple sclerosis treated with interferon beta. Before treatment, thyroid function tests were normal and anti-thyroid (anti-thyroglobulin and anti-thyroid peroxidase) antibodies were negative. After 18 months of treatment, the patient presented clinical features of thyroiditis. We noted a rise in TSH values and anti-thyroid antibodies were found positive. Treatment was not discontinued and further surveillance showed an improvement in clinical and biological features. We conclude that thyroid function should be monitored during INF beta treatment. Laboratory tests must be carried out before therapy. Clinical follow-up is indicated before undertaking biological tests.

Nigrostriatal dopaminergic depletion produces orofacial static mechanical allodynia.

BACKGROUND: Dopamine is implicated in different orofacial pain-related diseases. The mechanisms underlying this invalidating pain are not yet understood. Therefore, the present study investigated if unilateral or bilateral lesions of the medial forebrain bundle (MFB) could induce a trigeminal static mechanical allodynia (SMA) comparable to that obtained after chronic construction injury of the infraorbital nerve (CCI-IoN) in rats. METHODS: Unilateral and bilateral nigrostriatal lesions were obtained by injecting 6-hydroxydopamine into the MFB, and peripheral lesion was obtained by CCI-IoN. Static allodynia behaviour was tested by a mild non-noxious static von Frey filament stimulus. The analgesic effects of bromocriptine (D2R agonist) were assessed by both intraperitoneal and intracisternal injections. Finally, immunohistochemical study was done to investigate the implication of the protein kinase c isoform gamma (PKCγ) and the phosphorylated form of extracellular signal-related kinase 1/2 (pERK1/2) in pain sensitization at segmental level. RESULTS: 6-OHDA-lesioned animals developed SMA in the orofacial region as assessed by non-noxious stimuli. Intraperitoneal and intracisternal injections of bromocriptine alleviated this allodynic behaviour. Investigations within the medullary dorsal horn revealed an increase in PKCγ expression, a protein implicated in the chronicity of pain, within superficial laminae in 6-OHDA-lesioned rats. Also static mechanical stimulations of the orofacial region evoked increased expression of the molecular pain marker pERK1/2 in 6-OHDA-lesioned rats. CONCLUSION: Our data show that unilateral and bilateral dopamine depletion promoted trigeminal SMA comparable to that obtained after CCI-IoN. This allodynia can be alleviated by D2R activation, making D2R agonist a potential analgesic for orofacial neuropathic pain.

Localization and developmental expression of BK channels in mammalian cochlear hair cells.

The expression of Slo channels (alpha subunits of BK channels) was investigated in the developing mouse cochlea using a polyclonal antibody against the C-terminal part of the protein (residues 1098-1196). The first BK channel immunoreactivity was observed in the cochlea at E18, where it was localized within the cytoplasm of cells lining the area of the organ of Corti and the spiral ganglion. There was an increase of immunoreactivity in all cells bordering the scala media (supporting and hair cells of the organ of Corti, the stria vascularis and the Reissner's membrane) in the following stages (postnatal day [P] 0 and P6). From P12 to adult, a strong membranous labeling, increasing with age, appeared in inner hair cells. The distribution of BK channels was mainly observed as dense elongated plaques localized in the lateral membrane below the cuticular plate. In addition, a more discrete immunolabeling for BK channels, as punctuated dots, was observed in the synaptic area of inner hair cells. This dual localization of BK channels within inner hair cells was confirmed by a different technique using a fluorescently labeled high-affinity ligand of these channels: IbTX-D19C-Alexa488. We demonstrated under patch clamp experiments that this fluorescent toxin conserved its native property, i.e. to reversibly inhibit BK currents in isolated inner hair cells. The fluorescent toxin, both in living or fixed tissues, also showed a preferential binding to mature inner hair cells with a similar subcellular distribution described above using immunocytochemical technique. Overall, our present results confirm the appearance of membranous BK channels around P12 in mouse inner hair cells, an age at which the auditory system becomes functional. The expression of BK channels in mature inner hair cells, near the site of mechanical-transduction, might serve to limit receptor potential attenuation due to the space constant, and thus permitting these sensory cells to function as fast and sensitive transducers.

Mechanism of GABA involvement in post-traumatic trigeminal neuropathic pain: activation of neuronal circuitry composed of PKCγ interneurons and pERK1/2 expressing neurons.

BACKGROUND: GABA disinhibition within the spinal dorsal horn has been implicated in pain hypersensitivity on injury in different neuropathic models. However, GABA alteration has been explored in only one study on trigeminal neuropathic pain. METHODS: The present study investigated the implication of GABA in trigeminal dynamic mechanical allodynia (DMA) obtained after chronic constriction of the infraorbital nerve (CCI-IoN), and explored the cellular and molecular mechanisms by which GABA dysfunction induced DMA. RESULTS: Our data demonstrated a significant decrease in labelling in two GABA cell markers, glutamate acid decarboxylase (GAD67), and parvalbumin, in the medullary dorsal horn (MDH) of allodynic rats in comparison to sham rats. Increasing GABA by intracisternal injections of vigabatrin (VGB), a blocker of the catabolic enzyme GABA transaminase, alleviated pain behaviour and restored normal GABA cell marker expression in allodynic MDH. Interestingly, intracisternal VGB administration also significantly decreased PKCγ staining, i.e., of its phosphorylated active form and the number of pERK1/2 positive cells within the MDH. These two markers were highly expressed in allodynic MDH. CONCLUSION: The circuitry composed of PKCγ and pERK1/2 cells is silent under physiological conditions but is activated after CCI-IoN, therefore, switching touch stimuli to pain sensation. The decrease of GABA transmission constituted a key factor in the activation of this neuronal circuitry, which opens the gate for non-noxious stimuli to reach nociceptive projection neurons in lamina I.

Potential of some autochthonous wild plants of Burundi for vegetable oil and valuable compounds production / Potencial de algumas plantas silvestres autóctones do Burundi para a produção de óleos vegetais e compostos valiosos

Abstract Twelve species of indigenous plants have been studied in order to valorize some natural resources of Burundi (Eastern Africa) to investigate possibilities of vegetable oil production. Physicochemical properties and oil contents were determined from seeds harvested through five ecogeographic zones. From oilcake extracts, total sugars contents, proteins (TPrC), polyphenolic (TPhC), and flavonoids were quantified using spectrophotometry. Furthermore, antioxidant activity of oilcake extracts was assessed by 2, 2-diphenyl-b-picrylhydrazyl (DPPH) radical-scavenging and ferric reducing antioxidant power (FRAP) assays. All oil contents obtained were found to be quite similar to those of common oleaginous seeds. The two highest were found in Parinari curatellifolia (61.44 ± 4.81% Dry Matter) and Myrianthus arboreus (48.26 ± 5.96% DM). More than half of the species have shown TPrC ranging from 10 to 24% dry matter of oilcake (DM). Brachystegia longifolia was revealed exceptionally stronger antioxidant potential: effectiveness antiradical of 163.06 ± 26.29 mL/μg.min (DPPH assay) and reducing power of 2618.21 ± 161.22 GAE/100 g DM (FRAP assay). TPhC were positively correlated (p < 0.05) to the antioxidant activity. This pioneering work on these wild species highlight the potential for producing vegetable oil and valuable biomolecule sources likely for food, cosmetics, pharmacy and industry.

Resumo Doze espécies de plantas indígenas foram estudadas para valorizar alguns recursos naturais do Burundi (África Oriental), para investigar as possibilidades de produção de óleo vegetal. As propriedades físico-químicas e o conteúdo de óleo foram determinados com base em sementes colhidas em cinco zonas ecogeográficas. A partir de extratos de bagaço de óleo, os teores de açúcares totais, proteínas (TPrC), polifenólicos (TPhC) e flavonoides foram quantificados por espectrofotometria. Além disso, a atividade antioxidante dos extratos de bagaços foi avaliada por ensaios de 2,2-difenil-b-picrilhidrazil (DPPH) e antioxidante redutor de ferro (FRAP). Todos os conteúdos de óleo obtidos foram encontrados para ser bastante semelhantes aos das sementes oleaginosas comuns. Os dois maiores foram encontrados em Parinari curatellifolia (61,44 ± 4,81% de matéria seca [MS]) e Myrianthus arboreus (48,26 ± 5,96% de MS). Mais da metade das espécies mostrou TPrC variando de 10% a 24% de MS de tortas. Brachystegia longifolia revelou um potencial antioxidante excepcionalmente mais forte: eficácia antirradical de 163,06 ± 26,29 mL/μg.min (DPPH assay) e poder redutor de 2.618,21 ± 161,22 GAE/100 g de MS (ensaio FRAP). TPhC correlacionaram-se positivamente (p < 0,05) com a atividade antioxidante. Este trabalho pioneiro sobre essas espécies selvagens destaca o potencial para a produção de óleo vegetal e fontes valiosas de biomoléculas para alimentos, cosméticos, farmácia e indústria.

Cochlear innervation in the developing rat: an immunocytochemical study of neurofilament and spectrin proteins.

We have studied the innervation of the developing cochlea by immunocytochemical staining of the cytoskeletal proteins, neurofilament (NF), and spectrin (brain spectrin and erythrocyte spectrin). NF immunoreactivity was seen in spiral ganglion cell bodies and their processes and in fibers of the intraganglionic spiral bundle (IGSB) on gestational day 16. NF immunoreactivity with monoclonal antibodies to NF160 and NF68 was present beneath both inner hair cells (the IHC) and outer hair cells (OHCs) on gestational day 20. NF200 immunostaining was located only in the IGSB and in fibers reaching the IHC. The first NF200 immunoreactivity beneath the OHCs was seen in the basal turn at birth. NF labelling began to decrease on postnatal day 9 and its intensity became more like that of the adult. Brain spectrin immunostaining was first seen in the IGSB of the basal turn on gestational day 18. It reached the fibers between the spiral ganglion and the IHC on gestational day 20. Brain spectrin immunoreactivity was first seen beneath the OHCs in the basal turn at birth. It reached all the OHCs of the cochlea by postnatal day 4, and began to decrease 9 days after birth. Erythrocyte spectrin immunostaining was first observed during the second postnatal week, when it labelled spiral ganglion cells. The distribution of NF200 and brain spectrin immunoreactivity suggested that efferent innervation of OHCs is present at birth in the rat, and confirms previous studies showing the early efferent innervation of the OHCs of the mouse and the rat at birth, and the time lag between the appearance of the two spectrin isoforms during development.

Regional distribution of neurotrophin receptors in the developing auditory brainstem.

Neuron survival and axonal regeneration become severely limited during early postnatal development. In conjunction with our recent organotypic analysis of regeneration in the auditory midbrain, we wished to determine whether neurotrophins could serve as a trophic substance during the postnatal period. Therefore, the current study examines the development of three neurotrophin receptor tyrosine kinases (TrkA, TrkB, and TrkC) in the gerbil auditory brainstem. Immunoreactivity to TrkA, the nerve growth-factor receptor, was observed in nonneuronal cells during the first two postnatal weeks. In the cochlear nucleus of mature animals, however, there was a TrkA-positive neuronal subpopulation. In contrast, immunoreactivity to TrkB and TrkC (the receptors for brain-derived neurotrophic factor and neurotrophin-3, respectively) displayed a widespread distribution in the auditory brainstem. At postnatal day 0, TrkB and TrkC staining was virtually absent from auditory nuclei, although immunopositive neurons were present in the mesencephalic trigeminal nucleus. By postnatal day 7, TrkB- and TrkC-positive neurons were present in most brainstem auditory nuclei. At postnatal day 15, TrkB immunoreactivity was observed throughout the inferior colliculus (IC), the cochlear nucleus, the medial and lateral nuclei of the trapezoid body, and the lateral superior olive, whereas TrkC labeled only a subpopulation of neurons within the central nucleus of the IC. The TrkB immunoreactivity was present on both neuronal somata and dendrites, whereas TrkC was generally restricted to cell bodies. At postnatal day 30, TrkB immunostaining was observed on most neurons of the IC. The medial and lateral nuclei of the trapezoid body displayed extremely strong TrkB staining, followed by the cochlear nucleus. In contrast, the TrkC immunostaining was decreased dramatically by postnatal day 21. Observations at the ultrastructural level confirmed a neuronal localization of TrkB and TrkC. Immunostaining for both receptors was restricted largely to the postsynaptic density of synaptic profiles in both dendrites and somata. In summary, this study illustrates a differential pattern of immunoreactivity between three neurotrophin receptors during development. The general increase of TrkB expression is well correlated with the onset of sound-evoked activity in this system, and its synaptic localization suggests that it may be involved in the modulation or maintenance of postsynaptic physiology.

Developmental differentiation of MAP2 expression in the central versus the peripheral and efferent projections of the inner ear.

The goal of this study was to extend our knowledge of MAP2 localization in the peripheral nervous system of mammals, since most results on MAP2 distribution are obtained in the central nervous system (CNS). This study shows the presence of microtubule-associated protein 2b (MAP2b) and MAP2c in the inner ear and describes the immunocytochemical distribution of MAP in adult and developing spiral ganglion of the rat by using a well-characterized antibody for MAP2a and MAP2b. (This antibody does not recognize the immature MAP2c). MAP2 labeling is already present in spiral ganglion neurons at 16 days of gestation. From this stage and up to the first postnatal week, MAP2 labeling was strong in all spiral ganglion neurons and their central processes. Double immunostaining at the 16-day stage with anti-MAP2 and anti-neurofilament (NF) antibodies mainly showed NF labeling in central branches that corresponded to anatomically and functionally described axons of spiral neurons. The peripheral branches lacked MAP2 labeling. In neonatal and postnatal stages, MAP2 reactivity was located in spiral ganglion perikarya and their neurites. The intensity of adult labeling was, however, lower than in younger animals. The antibody used in this study did not label axons originating in the CNS as seen by a negative response in efferent fibers from the intraganglionic spiral bundle of the cochlea. Our results suggest that during ontogenesis, MAP2 is highly expressed in the central projection of spiral ganglion neurons, and then is reduced to lower quantities in the central branch after the first postnatal week and persists into adulthood.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of glutamate receptors GluR 2/3 and NR1 in the developing rat cerebellum.

The distribution of glutamate receptors GluR2/3 and NR1 was analysed immunohistochemically during development of the rat cerebellum. GluR2/3 immunoreactivity appeared by postnatal day P0 in somata of Purkinje cells. Throughout P7, P15, P20 and adulthood, GluR2/3 immunoreactivity was found in the entire Purkinje cell dendritic arbor reaching to the external granular layer and, by P15, the surface of the cerebellum. By P7, the granular layer revealed scattered, mildly reactive, cells. NR-1 immunoreactivity first gained prominence about P7 in the region of the multi-layered Purkinje cell somata. By P15, NR1 was prominent in Purkinje cell somata and Golgi cells. The reaction product extended into the primary main dendrite of Purkinje cells. By P21, stellate and basket cells had intense reactivity throughout the molecular layer and reactive large-diameter dendrites of Golgi cells projected toward the molecular layer. Granule cells remained very weak among strongly reactive Golgi cell somata and dendrites. Ultrastructural immunohistochemistry revealed NR1 reaction product in Purkinje cell somata, in stellate cell somata and dendrites and on postsynaptic membranes of scattered spines throughout the molecular layer. The later appearance and restricted location of NR1 in somata and proximal dendrites of Purkinje cells contrasted markedly with GluR2/3 which appeared before birth and remained prominent throughout Purkinje cell dendritic arbors of adults. The time of NR1 expression correlated with the generation of granule cells, their synaptogenesis on Purkinje cells, the formation of stellate/baske cells and the shift of climbing fibre synapses from distal to proximal dendrites. The developmental appearance of stellate/basket cells and Golgi cells as well as their high reactivity remaining into adulthood suggest that these inhibitory molecular and granular layer interneurons are the principal targets of glutamate axons serving NR1 synaptic properties while Purkinje cells and brush type granule cells are targets for glutamate connections with GluR2/3 characteristics.

Structural and molecular heterogeneity of astrocytes and oligodendrocytes in the gerbil lateral superior olive.

The goal of this study was to determine the distribution and diversity of astrocytes and oligodendrocytes within the lateral superior olive of the gerbil. We used morphometric analyses and several immunocytochemical markers to assess differences in glial cell composition between the lateral (low-frequency projection) and the medial (high-frequency projection) limb of the lateral superior olive. Cell counts from Toluidine-stained semithin sections revealed a similar density of total astrocytes in both the lateral and the medial limbs. However, based on cytologic features, there was a prevalence of fibrous-like astrocytes in the lateral limb and protoplasmic-like astrocytes in the medial limb. In a similar manner, glial fibrillary acidic protein staining of astrocytes was intense in the lateral limb, but was largely restricted to the nucleus borders in the medial limb of the lateral superior olive. While glial fibrillary acidic protein was largely restricted to astrocytic processes, glutamine synthetase and S100 protein staining occurred, for the most part, in glial cell bodies. The density of glutamine synthetase positive cell bodies was homogeneous between the two limbs, while the density of S100-positive somata was significantly greater in the lateral limb. Cell counts obtained from semithin sections demonstrated a greater density of oligodendrocytes in the lateral limb than in the medial limb of the lateral superior olive. In a similar manner, there was a 40% greater density of carbonic anhydrase-positive somata in the lateral limb compared to the medial limb. Transferrin immunostaining was restricted to oligodendrocytes, but the density of labeled somata was identical in the lateral and medial limbs. 2',3'-Cyclic nucleotide 3'-phosphodiesterase and myelin-associated glycoprotein were also localized to the somata of oligodendrocytes, labeling both perisomatic and interfascicular cells. At the ultrastructural level, specialized contacts were found between pairs or clusters of oligodendrocytes. These results suggest that more than one type of astrocyte and oligodendrocyte is present within the gerbil lateral superior olive. Furthermore, glial cells were unevenly distributed, such that a greater density of oligodendrocytes and fibrous-like astrocytes were found in the low-frequency projection region. This heterogeneity is well correlated with known differences in the neuronal morphology within the lateral superior olive.

Peripherin-like immunoreactivity in type II spiral ganglion cell body and projections.

Peripherin, an intermediate filament protein, is present in neuronal subpopulations of both peripheral and central nervous systems. The distribution of peripherin was studied in the adult rat cochlea using immunohistochemistry on whole mount material, in cryostat sections and sections of plastic embedded tissue. In the spiral ganglion, peripherin labeling was restricted to the perikarya of a subpopulation of neurons and their peripheral and central processes. Peripherin positive neurons had the following features: (i) they have a large eccentric nucleus, they were often found in a cluster of 2 or 3 cells, (ii) they were often located near the intraganglionic spiral bundle fibers, (iii) they represented roughly 8% of the whole ganglion population and (iv) on the average they had smaller perikarya than non-immunoreactive cells. Immunostaining on semithin plastic sections revealed positive reactivity on Type II ganglion cells, while Type I neurons were negative. Double labeling using peripherin and three neurofilament (NF) subunit antibodies confirmed the presence of both markers within the same spiral ganglion cell type. Type II neurons have been previously documented as the only subpopulation of the spiral ganglion that presents a strong positive NF immunoreactivity within their perikarya. In the organ of Corti, peripherin-positive fibers formed bundles that course beneath the outer hair cells and send branches that end as boutons contacting the outer hair cells. All these characteristics suggest that peripherin-positive cells are Type II neurons, and that peripherin constitutes a reliable marker for this spiral ganglion subpopulation, as well as their peripheral and central processes.

Immunocytochemical localization of neurofilament protein subunits in the spiral ganglion of the adult rat.

Spiral ganglion neurons from adult rats were treated with several monoclonal antibodies that react with neurofilaments (NFs) and NF subunits. An antibody against NFs used with immunocytochemical techniques showed a strong reaction with most neuron processes in the spiral ganglion, whereas only a few neurons presented a reaction. Using monoclonal antibodies against the 3 subunits, we obtained the same results with a small percentage of neurons labelled. From quantitative observations, reacting neurons showed the same percentage as and a smaller size than T II neurons observed with a more conventional method. This shows that reacting neurons are indeed T II neurons and that they can easily be differentiated by an accumulation of NFs in their perikaryon by well characterized commercially available antibodies.

Differential expression of MAG, MBP and L1 in the developing lateral superior olive.

The aim of this study was to investigate whether glial-associated molecules exhibit a pattern of expression that could influence oriented dendrite outgrowth in the gerbil lateral superior olive (LSO). In particular, we have previously noted that axon fascicles are oriented parallel to isofrequency laminae in the medial limb of the LSO, as are LSO dendrites, a phenotype that emerges postnatally. Therefore, we examined the immunocytochemical staining pattern of antibodies directed against three proteins that are found along axons: myelin basic protein (MBP), myelin-associated glycoprotein (MAG), and neuron-glia cell adhesion molecule (L1). MAG staining was first observed at postnatal day (P) 4 on the axon fibers surrounding the LSO. By P7 there was a differential pattern of MAG staining within the LSO, and immunopositive fibers were observed solely in the medial limb (e.g., high frequency projection region). Between P7 and P12, MAG staining was restricted largely to fascicles in the medial limb, and these were oriented parallel to the isofrequency axes. Few positive fibers of irregular orientation were observed in the lateral limb (e.g., low frequency projection region). Significant MAG-staining was not observed in the lateral limb until P15. The MAG immunoreactivity extended throughout the LSO by P21, although it was no longer restricted to axon fascicles. In contrast, MBP-positive fibers were uniformly distributed within the LSO by P12. Finally, L1 was found on oriented axon fascicles at P0, but became sparsely distributed throughout the LSO neuropil after P7, and was restricted to neuron cell bodies in the adult. Taken together, the results suggest that oriented axon fascicles bearing MAG and L1 may contribute to the developmental refinement of dendrite and axon arbors within the LSO.

Distribution of BDNF, NT-3 and NT-4 in the developing auditory brainstem.

This study describes the developmental expression of three neurotrophins, brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3) and neurotrophin (NT-4) in the rat auditory brain-stem using immunohistochemistry. At postnatal day 0 (PND 0), neurotrophins expression was virtually absent from all auditory nuclei in the brainstem, even though some positive neurons were observed in the mesencephalic trigeminal nucleus at this age. However, BDNF, NT-3 and NT-4 positive neurons were observed in most brainstem auditory nuclei by PND 6. At the following stages, there was a general increase in the intensity of the neurotrophins immunoreactivity and BDNF labeling was particularly prominent in most cochlear nucleus neurons. A differential pattern of staining emerged in cochlear nucleus subdivisions, with more intense staining present in the ventral part. The superior olivary complex nuclei followed a similar pattern of BDNF staining compared to the cochlear nucleus. In the adult, BDNF heavily labeled most neurons of the superior olivary nuclei and moderately labeled neurons of the inferior colliculus (IC). NT-3 and NT-4 showed a similar pattern of staining in most auditory brainstem nuclei. The first staining was observed by PND 6 in some neuronal cell bodies. NT-3 and NT-4 immunoreactivity increased in the following stages and in the adult moderate labelings were observed in most neurons of the cochlear nucleus, the superior olivary nuclei and the IC. These results show that neurotrophins are expressed 1 week before the onset of hearing and the increase of their expressions correlate with the appearance of sound-evoked activity in the system. The temporal distribution of neurotrophins does not correlate with neuronal birth, axonal outgrowth or the formation of connection in the auditory structures, suggesting a role primarily in the maintenance and/ or modulation of postnatal and adult functions.

Ontogenesis of type II spiral ganglion neurons during development: peripherin immunohistochemistry.

In this study, we analysed the distribution of the intermediate filament peripherin in the developing cochlea of the rat. At gestational day 16, weak immunolabeling was observed in neuronal somas throughout the spiral ganglion. At gestational day 20, the peripherin labeling increased in intensity throughout the spiral ganglion. At gestational day 20, the peripherin labeling increased in intensity throughout the cochlea but became especially strong in some ganglion neurons of the basal turn. Homogeneous immunolabeling was observed throughout the spiral ganglion of the apical turn. Double immunofluorescence labeling of the prenatal cochlea with peripherin and neurofilament (NF) antibodies revealed colocalization on the same structures. By postnatal day 3, the peripherin labeling intensity had decreased in the majority of spiral ganglion neurons, but remained strong in some cells of the basal turn. Only a few neurons continued to be immunolabeled into adulthood that correspond to Type II spiral ganglion neurons expressing both NF protein and peripherin, two classes of intermediate filament proteins. In the organ of Corti, the first immunolabeling was observed on gestational day 20 as peripheral fibers reaching the receptor cells. Positive fibers were observed below both inner (IHCs) and outer (OHCs) hair cells. At birth and at postnatal day 3, peripherin immunolabeling was still observed below both IHCs and OHCs. By postnatal day 4, peripherin labeling became more dominant in fibers below OHCs, but some immunoreactivity was still present below IHCs. No immunoreactivity was present in the intraganglionic spiral bundle (IGSB) fibers containing the olivary complex efferent fibers before birth. A few days after birth some fibers of the IGSB started to be immunoreactive.

First appearance of type II neurons during ontogenesis in the spiral ganglion of the rat. An immunocytochemical study.

Ontogenesis of spiral ganglion in the rat was studied using antibodies to three subunits of neurofilaments (NFs): NF 68 KDa, NF 160 KDa and NF 200 KDa. The expression of immunoreactivity was examined with 3 immunocytochemical methods: indirect immunofluorescence, peroxidase-antiperoxidase and avidin-biotin complex. Aim of the study was to detect the time of differentiation of the spiral ganglion type II neurons. At 16 and 18 days of gestation, most neuron cell bodies express immunoreactivity to only two NF subunits: NF 68 and NF 160, but at birth they react with the antibodies to all 3 subunits albeit weakly. Nevertheless, a small population (about 7%) of nerve cells that strongly reacts against all 3 NF subunits emerges in the basal turn, already at 20 days of gestation. Two to 3 days after birth, the strongly stained cells are dispersed throughout the entire ganglion. The intensity of their reaction to the NF antibodies is similar to that seen in the adult animal. The strong immunoreactivity of this selective neuronal population suggest, that they correspond to the type II spiral ganglion neurons. Our results imply that the differentiation between the type I and the type II of spiral neurons in the rat occurs perinatally.

Developmental expression of Ca(v)1.3 (alpha1d) calcium channels in the mouse inner ear.

Voltage-gated calcium channels are important for neurotransmission at the level of inner hair cells (IHCs) and outer hair cells (OHCs). These channels open when mechanical stimulation depolarises the hair cell membrane and the resulting calcium influx triggers neurotransmitter release. Voltage-gated calcium channels expressed in hair cells are known to be of the L-type with a predominance of the Ca(v)1.3 subunit. The present study describes the developmental expression of the Ca(v)1.3 protein in the cochlea and the vestibular system using immunohistochemical technique. In the adult organ of Corti (OC), Ca(v)1.3 was localized in both sensory and non-sensory cells with a more intense expression in IHCs and Deiters cells when compared to OHCs. In both hair cell types, immunoreactivity was observed in the apical pole, basolateral membrane and at the basal pole (synaptic zone). Similar results were obtained in the vestibular organs. During development, Ca(v)1.3 immunoreactivity was observed in the cochlea as early as embryonic day 15, with expression increasing at birth. At these early stages of cochlear development, Ca(v)1.3 was expressed in all cell types surrounding the scala media. In the OC, the labeling was observed in IHCs, OHCs and supporting cells. The Ca(v)1.3 expression reached an adult-like pattern by the end of the second postnatal week. The present findings suggested that, in addition to their implication in hair cells synaptic transmission, Ca(v)1.3 calcium channels also play an important role in vesicle recycling and transport, as suggested by their extrasynaptic location at the apical pole of the hair cells. The Ca(v)1.3 channels in Deiters cells could participate in active calcium-induced changes in micromechanics of these supporting cells. An early expression during development suggested that these calcium channels are in addition important in the development of the cochlear and vestibular sensory epithelium.

Neurofilament immunoreactivity in vestibular ganglion neurons of the adult rat.

Immunocytochemical methods were used to study the distribution of neurofilament (NF) proteins in vestibular ganglion neurons of the adult rat. Monoclonal antibodies against the three triplet proteins were used. By indirect immunofluorescence and the peroxidase-antiperoxidase method, two populations of neurons were distinguished. One population with large perikarya showed strong NF immunoreactivity. A second population of neurons presented only slight or no immunoreactivity. The strong NF immunoreactivity in the perikarya of certain neurons seems to be a general feature of many sensory ganglia.