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.

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.

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

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/g.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/g.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.

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.

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.

Astrocytes are involved in trigeminal dynamic mechanical allodynia: potential role of D-serine.

Trigeminal neuropathic pain affects millions of people worldwide. Despite decades of study on the neuronal processing of pain, mechanisms underlying enhanced pain states after injury remain unclear. N-methyl-D-aspartate (NMDA) receptor-dependent changes play a critical role in triggering central sensitization in neuropathic pain. These receptors are regulated at the glycine site through a mandatory endogenous co-agonist D-serine, which is synthesized by astrocytes. Therefore, the present study was carried out to determine whether astrocytes are involved, through D-serine secretion, in dynamic mechanical allodynia (DMA) obtained after chronic constriction of the infraorbital nerve (CCI-IoN) in rats. Two weeks after CCI-IoN, an important reaction of astrocytes was present in the medullary dorsal horn (MDH), as revealed by an up-regulation of glial fibrillary acidic protein (GFAP) in allodynic rats. In parallel, an increase in D-serine synthesis, which co-localized with its synthesis enzyme serine racemase, was strictly observed in astrocytes. Blocking astrocyte metabolism by intracisternal delivery of fluorocitrate alleviated DMA. Furthermore, the administration of D-amino-acid oxidase (DAAO), a D-serine-degrading enzyme, or that of L-serine O-sulfate (LSOS), a serine racemase inhibitor, significantly decreased pain behavior in allodynic rats. These results demonstrate that astrocytes are involved in the modulation of orofacial post-traumatic neuropathic pain via the release of the gliotransmitter D-serine.

Low cost biosorbent "banana peel" for the removal of phenolic compounds from olive mill wastewater: kinetic and equilibrium studies.

The aim of this work is to determine the potential of application of banana peel as a biosorbent for removing phenolic compounds from olive mill wastewaters. The effect of adsorbent dosage, pH and contact time were investigated. The results showed that the increase in the banana peel dosage from 10 to 30 g/L significantly increased the phenolic compounds adsorption rates from 60 to 88%. Increase in the pH to above neutrality resulted in the increase in the phenolic compounds adsorption capacity. The adsorption process was fast, and it reached equilibrium in 3-h contact time. The Freundlich and Langmuir adsorption models were used for mathematical description of the adsorption equilibrium and it was found that experimental data fitted very well to both Freundlich and Langmuir models. Batch adsorption models, based on the assumption of the pseudo-first-order, pseudo-second-order and intraparticle diffusion mechanism, showed that kinetic data follow closely the pseudo-second-order than the pseudo-first-order and intraparticle diffusion. Desorption studies showed that low pH value was efficient for desorption of phenolic compounds. These results indicate clearly the efficiency of banana peel as a low-cost solution for olive mill wastewaters treatment and give some preliminary elements for the comprehension of the interactions between banana peel as a bioadsorbent and the very polluting compounds from the olive oil 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.

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.

CNTFRalpha and CNTF expressions in the auditory brainstem: light and electron microscopy study.

CNTF receptor alpha (CNTFRalpha) is involved in the development, the maintenance and the regeneration of a variety of brain structures. However, its in vivo distribution has not been determined in the auditory system. CNTFRalpha expression was studied in developing and adult rat brainstem auditory nuclei using immunohistochemistry. At birth, the CNTFRalpha immunolabeling was clearly present in somata of the external nucleus of the inferior colliculus but was diffuse throughout brainstem auditory nuclei. The labeling was present in most brainstem auditory nuclei by post-natal day (PND) 6. The intensity of the staining subsequently increased to its highest level at PND21 and decreased to an adult-like appearance by the fourth post-natal week. In adult, CNTFRalpha labeling occurred in most neurons of the cochlear nucleus (CN), the lateral superior olive (LSO), the medial superior olive (MSO), and the medial nucleus of the trapezoid body (MNTB). CNTFRalpha labeling first appeared in the central nucleus of the inferior colliculus (IC) by the end of the fourth week. There was a general increase in the expression of CNTFRalpha that begins prior to the onset of hearing and reaches its highest level after this important developmental stage. Ultrastructural analysis in the adult ventral CN revealed the presence of CNTFR in post-synaptic sites. The presence of CNTF has been investigated in the adult using both Western blot and immunohistochemistry. Western blot showed the presence of CNTF in both peripheral and central auditory structures. The CNTF label was generally localized to the somatic compartment, in axons and as puncta surrounding neuronal cell bodies and dendrites. Differential CNTF labeling was observed between the different auditory nuclei. CNTF staining is present in neurons of the CN, the MNTB and the LSO, while it is restricted to axons and puncta surrounding neuronal somata in the IC. The clear presence of CNTFRalpha at post-synaptic terminals and that of its ligand the CNTF in axons and puncta surrounding neuronal cell bodies suggest an anterograde mode of action for CNTF in the central auditory system.

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.

Age-dependent failure of axon regeneration in organotypic culture of gerbil auditory midbrain.

Inferior colliculus (IC) slice cultures from postnatal (P) day 6-8 gerbils exhibit axonal regeneration across a lesion site, and these regrowing processes can form synapses. To determine whether regenerative capacity is lost in older tissue, as occurs in vivo, slices from P12-21-day animals were grown under similar conditions. While these cultures displayed a near complete loss of neurons over 6 days in vitro, glutamate receptor antagonists (AP5 and/or CNQX) significantly enhanced survival, particularly at P12-15. In contrast, several growth factors or high potassium did not improve neuron survival. Therefore, axonal regeneration was assessed following complete transection of the commissure in AP5/CNQX-treated IC cultures from P12 animals. Neurofilament staining revealed that transected commissural axons survived for 6 days in vitro, but only a few processes crossed the lesion site and these axons did not extend into the contralateral lobe. In contrast, there was robust axonal sprouting and growth within one lobe of the IC, remote from the lesion site. When P6 and P12 tissue was explanted onto a coated substrate, the P6 axons grew onto the substrate, but the P12 axons were seemingly prevented from reaching the substrate by a veil of nonneuronal cells. Coculture of the IC and one of its afferent populations, the lateral superior olive, provided a similar finding, indicating that failure to regenerate was a general property at the age examined. These data show that neuron survival is not sufficient to permit axon regeneration at P12, and suggest that P12 lesion sites manufacture a prohibitive substrate since process outgrowth is blocked specifically at the commissure transection.

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.

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.

In vitro induction of microcyst-like structures in the superior olivary complex.

To investigate the etiology of hole formation in the gerbil and rat central auditory system, organotypic cultures were grown in control and veratridine-containing media. The latter condition is known to increase neuronal activity. Tissue was obtained at postnatal day 6 and grown for 6-9 days in vitro, a period prior to the formation of holes in vivo. In both rats and gerbils, veratridine led to the appearance of large numbers of holes, and these were phenotypically similar to those found in vivo. These results support the idea that hole formation is an activity-dependent phenomenon, and suggest that it is not restricted to the mature gerbil auditory system.

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.

Glycinergic transmission regulates dendrite size in organotypic culture.

We previously demonstrated that inhibitory synaptic transmission influences dendrite development in vivo. We now report an analogous finding in an organotypic culture of a glycinergic projection nucleus, the medial nucleus of the trapezoid body (MNTB), and its postsynaptic target, the lateral superior olive (LSO) of gerbils. Cultures were generated at 6-7 days postnatal and grown in serum containing medium with or without the glycine receptor antagonist, strychnine (SN), at 2 microM. LSO neurons were then labeled with biocytin, and the dendritic arbors were analyzed morphometrically. Compared to neurons form age-matched in vivo tissue, the neurons cultured in control media were somewhat atrophic, including decreases in dendritic branching and length. Incubation in strychnine led to a dramatic increase in dendritic branching and total dendritic length. Control neurons averaged 6.3 branches compared to 18 branches/neuron in SN-treated cultures. There was a similar increase in primary dendrites and total dendritic length. The physical elimination of MNTB cells did not mimic SN treatment, presumably because glycinergic LSO neurons generated intrinsic connections. In fact, the LSO soma area was significantly greater following MNTB removal, suggesting that these afferents provide a second signal to postsynaptic neurons. These results suggest that spontaneous glycinergic transmission regulates the growth of postsynaptic processes.

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.