Cyclic GMP evoked calcium transients in olfactory receptor cell growth cones.

Nitric oxide-induced calcium transients in growth cones are believed to be mediated by cyclic nucleotides. Because nitric oxide is thought to influence the development of olfactory receptor cells (ORCs), we have begun to explore the effect of cyclic nucleotides on ORC growth cones. Cultured ORCs were loaded with fluo-3 AM and confocal imaging was employed to monitor calcium transients following cyclic nucleotide-gated channel activation. Application of 8-bromo-cGMP at the growth cone caused transient increases in fluorescence which were restricted to the growth cone and lasted tens of seconds. The signal was abolished by LY83583, an inhibitor of cyclic nucleotide-gated channels. 8-Bromo-cGMP also inhibited further extension of growth cones. The data indicate that ORC growth cones exhibit cGMP-dependent calcium transients that are consistent with those generated by cyclic nucleotide-gated channels.

Neurotrophin-evoked rapid excitation through TrkB receptors.

Neurotrophins are a family of structurally related proteins that regulate the survival, differentiation and maintenance of function of different populations of peripheral and central neurons. They are also essential for modulating activity-dependent neuronal plasticity. Here we show that neurotrophins elicit action potentials in central neurons. Even at low concentrations, brain-derived neurotrophic factor (BDNF) excited neurons in the hippocampus, cortex and cerebellum. We found that BDNF and neurotrophin-4/5 depolarized neurons just as rapidly as the neurotransmitter glutamate, even at a more than thousand-fold lower concentration. Neurotrophin-3 produced much smaller responses, and nerve growth factor was ineffective. The neurotrophin-induced depolarization resulted from the activation of a sodium ion conductance which was reversibly blocked by K-252a, a protein kinase blocker which prefers tyrosine kinase Trk receptors. Our results demonstrate a very rapid excitatory action of neurotrophins, placing them among the most potent endogenous neuro-excitants in the mammalian central nervous system described so far.

Seasonal changes in astrocytes parallel neuronal plasticity in the song control area HVc of the canary.

In the song control area HVc of the canary, intercellular dye-coupling among astrocytes was studied by intracellular injection of neurobiotin into identified single astrocytes. Injection of individual astrocytes into acute slices resulted in dye spread to neighboring astrocytes, covering a sphere of up to 1 mm in diameter. The astrocytic nature of the dye-coupled cells was verified by double labeling of neurobiotin-filled cells with antisera for the astrocytic filament proteins GFAP or vimentin. The similarity in the number of dye-coupled cells and the total number of astrocytes labeled by immunocytochemical markers indicate that dye-coupling is specific for astrocytes and labels almost the entire local astrocytic population. Within the major nucleus for vocal control (HVc), approximately 25% more astroglial cells were present than in the surrounding forebrain tissue. There is no apparent hindrance of dye spread at the border of the HVc. The density of dye-coupled astrocytes and the expression of cytoskeletal filament proteins differed markedly between the reproductive period in spring and the quiescent period in autumn. While vimentin is the major astroglial filament in autumn, GFAP is strongly expressed in spring. The density of dye-coupled astrocytes reveals a marked increase in the reproductive period, followed by a reduction in autumn. The data indicate that the astrocytic population in the avian forebrain undergoes significant changes coincident with the known functional changes in the vocal control nuclei during periods of song production.

Olfactory ensheathing cells promote neurite extension from embryonic olfactory receptor cells in vitro.

The role of ensheathing cells, a macroglial cell type with a unique presence in the olfactory system, in the outgrowth of olfactory receptor cell neurites was explored in vitro. Glial cell cultures harvested from both the olfactory bulb nerve layer and the hippocampus were established and immunocytochemically characterized. The expression of the p75 low-affinity nerve growth factor receptor by ensheathing cells was used to distinguish them from other macroglial subpopulations. Results indicated that ensheathing cell cultures were approximately 80% pure. Olfactory receptor cells were cocultured with ensheathing or hippocampal glial cells or were seeded on laminin or poly-L-lysine as controls. Olfactory receptor cells extended the longest primary neurites when cocultured with ensheathing cells. Neurite extension on hippocampal glia and laminin was less extensive than that observed on ensheathing cells but higher than that on poly-L-lysine. The neurite outgrowth-promoting effect of ensheathing cells was, at least in part, mediated by diffusible factors, because olfactory receptor cell neurite extension could also be facilitated when receptor cells were cultured in ensheathing cell-conditioned media. In contrast, cortical neurons extended neurites of equivalent lengths on ensheathing and hippocampal glia. The results suggest that ensheathing cells may release factors that support the continuous outgrowth of olfactory receptor cell axons and, therefore, the capacity of this pathway to recover from injury.

The expression of tenascin-C along the lamprey olfactory pathway during embryonic development and following axotomy-induced replacement of the olfactory receptor neurons.

Extracellular guidance molecules affect the pathway of growing axons by both attractive and repulsive interactions. Tenascin-C, a glycoprotein of the extracellular matrix, is localized along developing axonal pathways where it may function by repulsion, restricting axons within specific boundaries. The lamprey olfactory pathway offers an advantageous model for studying the role of extracellular matrix proteins in axon guidance because the entire pathway is readily seen in horizontal sections and because lesioning the olfactory nerve will induce the system into a new phase of coordinated neurogenesis and axon outgrowth. Although tenascin-C expression was absent during embryonic development, olfactory nerve fascicles contained tenascin-C-immunoreactivity (IR) during the larval stage. During retrograde degeneration, the fascicles lost tenascin-C-IR. Diffuse unfasciculated axonal processes extending from the olfactory epithelium did not express tenascin-C-IR; however, acetylated tubulin and GAP-43-IR was present, indicating axonal outgrowth. When the newly extended axons of olfactory receptor neurons converged to form fascicles, tenascin-C-IR was evident within the fascicular boundaries. The absence of tenascin-C expression when axonal process were short and diffuse, and its return when axons coalesced within fascicles, supports the view that tenascin-C functions as a boundary molecule in the olfactory pathway.

Differential expression of extracellular matrix and cell adhesion molecules in the olfactory nerve and glomerular layers of adult rats.

Owing to the continual turnover of afferent input, the olfactory system offers a unique opportunity to study development and reorganization of neuronal networks in adults. To explore substrates that may underlie these processes in the adult olfactory system, we examined the expression and distribution of extracellular matrix and cell adhesion molecules (CAM) thought to be involved in axon guidance/extension. N-CAM, laminin, and tenascin were all detected by immunocytochemistry in the nerve and glomerular layers of the adult rat olfactory bulb, although the intensity and laminar distribution were varied. Antisera for N-CAM(total), N-CAM180, and tenascin bound to fascicles within the olfactory nerve layer and the glomerular neuropil. However, binding was nonuniform in that only subsets of axon fascicles and restricted glomeruli showed evidence of immunoreactivity. Antilaminin and a polyclonal antitenascin similarly exhibited heterogeneous intralaminar immunoreactivity. Tenascin colocalized with glial processes at the borders of glomeruli and subcompartments of the glomerular neuropil. Laminin immunoreactivity was evident in subsets of olfactory nerve fascicles and, to a lesser extent, the glomeruli. The data are consistent with the notion that ongoing axon extension and glomerular targeting in the olfactory system is subserved in part by a heterogeneous expression of the same extracellular matrix and CAMs present at higher levels during perinatal development.

The influence of ensheathing cells on olfactory receptor cell neurite outgrowth in vitro.

We previously reported that laminin substrates increased primary (1 degree) neurite outgrowth from olfactory receptor cells (ORCs) in vitro. To further explore mechanisms underlying the outgrowth of ORC neurites, we have cocultured ORCs with the ensheathing cells (ENSH) from the olfactory nerve. ORCs were plated either: (i) directly on monolayers of ENSH (prepared with minor modifications as reported by Doucette and Devon, or (ii) on coverslips suspended above the ENSH monolayer to investigate diffusible trophic influences of ENSH. In addition, ORCs were cocultured with either olfactory bulb glia (OBG) or hippocampal astrocytes (HG) or grown on either laminin (LN) substrates or poly-L-lysine (PLL) controls. The length of ORC neurites was determined after 48 hr in vitro. Immunocytochemical characterization of the ENSH cultures for p75 nerve growth factor (NGF) receptor and glial fibrillary acidic protein (GFAP) revealed that those cultures contained more than 80% ENSH. In OBG cultures approximately 10% and in HG cultures no cells with ENSH characteristics were found. All cells with ENSH characteristics were also LN-immunoreactive. After 48 hr in culture ORCs had the longest 1 degree neurites when they were cocultured with ENSH. No significant differences in the 1 degree neurite length were found comparing ORCs grown directly on ENSH and ORCs physically separated from ENSH. ORCs cultured on HG and on EHS-LN showed no significant differences in the ORC 1 degree neurite length, but on both substrates the ORC 1 degree neurites were significantly shorter than on ENSH. The length of the ORC secondary neurites did not vary significantly in the different culture conditions. Our results suggest that while LN appears to contribute to ORC neurite extension, additional diffusible factors released from ENSH are likely to be further determinants of neurite outgrowth. Because the OBG and HG cocultures did not influence ORC neurite outgrowth as significantly as did the ENSH, it seems plausible to suggest that these diffusible factors may be specific to subpopulations of glial cells.

Role of laminin in axonal extension from olfactory receptor cells.

The role of laminin, an extracellular matrix molecule believed to be involved in axon extension, was explored in the outgrowth of olfactory receptor cells and therefore in the maintenance of organization in the olfactory pathway. First, immunocytochemistry was used to examine laminin expression in the olfactory nerve and bulb during development. Laminin immunoreactivity was high in the olfactory nerve and glomerular layers. Although it declined in intensity, laminin expression continued in the nerve and in single glomeruli of adults. Second, the influence of laminin on neurite outgrowth was examined in vitro using olfactory receptor cells harvested from E14 rat embryos. We developed an in vitro assay to quantify the substrate preference of outgrowing neurites. Cells were cultured for 48 h on coverslips coated with either poly-L-lysine alone, or poly-L-lysine overlaid with laminin. On laminin-coated regions of coverslips, the primary neurites of olfactory receptor cells were 52% longer than on the poly-L-lysine control substrates. In addition, the direction of the neurite outgrowth was influenced by laminin. Fifty-six percent of all receptor cells located in a defined area surrounding a laminin zone extended neurites onto laminin. In contrast, only 7% of all receptor cells located in the corresponding laminin zone extended a neurite onto poly-L-lysine. In summary, these data suggest that laminin provides a favorable substrate for the extension of the primary neurite from olfactory receptor cells and the direction of their extension. Therefore, laminin may be a factor underlying continuous olfactory receptor cell axon outgrowth and its pathfinding in the olfactory system.

Testosterone elevates expression of tenascin-R and oligomannosidic carbohydrates in developing male zebra finches.

The song system of zebra finches is a model for studying the influence of steroids on neural connectivity and behavior during development. To investigate the molecular mechanisms underlying the song-related and gonadal hormone-regulated development of neural activity, we have chosen to investigate the expression of recognition molecules in the brain nuclei associated with motor control of song production. Here we show that testosterone accelerates expression of the predominantly oligodendroglia-, but also neuron-associated extracellular matrix glycoprotein tenascin-R and the oligomannosidic carbohydrate L3 during the third and seventh posthatching week in the higher vocal center (HVC) and robust nucleus of the archistriatum (RA), but not in other brain regions. The results suggest that recognition molecules and associated carbohydrate structures can be regulated by testosterone and that an increased expression of these molecules correlates with testosterone-induced modifications of song behavior.

Exogenous testosterone alters expression pattern of cell recognition molecules in brains of juvenile zebra finches.

We assessed spatio-temporal expression of tenascin and janusin by immunocytochemistry in testosterone-treated zebra finches and in untreated controls. These cell recognition molecules and components of the extracellular matrix mediate neurone-glia interactions, which are crucial for differentiation of ordered neural structures. We describe boundaries for the spatio-temporal expression of cell recognition molecules that highly correlate with the song motor centres as defined by combined neuroanatomical and ethological studies on song vocalization. The expression of janusin is accelerated by exogenous testosterone.

Antioestrogen inhibits myelination in brains of juvenile Zebra finches.

The effects of the antioestrogen Keoxifene on the ontogenetic process of myelination, on the differentiation of neurones of telencephalic song motor centres, on cerebellar structures and on behaviour were studied in male Zebra finches. Brain differentiation was studied by using computer aided analysis of silver impregnated brain sections and by measuring soma sizes of neurones after Nissl staining. An antioestrogen induced inhibition of myelination could be detected in the song motor centre robust nucleus of the archistriatum (RA) and in the cerebellum, whereas the region magnocellular nucleus of the anterior neostriatum (MAN) showed no difference between treated birds and controls.

Application of testosterone accelerates oligodendrocyte maturation in brains of zebra finches.

The effects of exogenously applied hormones on glial cell maturation of telencephalic song motor centers, midbrain and cerebellar structures was studied in juvenile male zebra finches. Testosterone was administered and the development of oligodendrocytes was studied using immunochemistry and computer aided image analysis on silver impregnated brain sections. A testosterone induced acceleration of oligodendrocyte maturation could be detected in several brain areas by using the monoclonal antibody O 10 recognizing an oligodendrocyte-specific cell surface antigen and by the silver impregnation for myelin. The increase in myelin density was higher in the testosterone treated animals than in the control animals in the forebrain and in the cerebellum, whereas two regions in the midbrain showed no difference between treated and controls.