CNS glial cells support in vitro survival, division, and differentiation of dissociated olfactory neuronal progenitor cells.

Olfactory receptor neurons (ORNs) are replaced and differentiate in adult animals, but differentiation in dissociated cell culture has not been demonstrated. To test whether contact with the CNS regulates maturation, neonatal rat olfactory cells were grown on a culture substrate or on CNS astrocytes. Mature ORNs, immunopositive for olfactory marker protein (OMP), disappeared rapidly from both systems. Neurons positive for neuron-specific tubulin (immature and mature) disappeared from substrate-only cultures, but remained abundant in the cocultures. OMP-positive neurons reappeared after 10 days in vitro. Pulse labeling with [3H]thymidine showed extensive neurogenesis of both immature and mature olfactory neurons. This demonstrates, in vitro, both division and differentiation of olfactory progenitor cells.

Initial observations on using magnesium metal in peripheral nerve repair.

Biodegradable magnesium metal filaments placed inside biodegradable nerve conduits might provide the physical guidance support needed to improve the rate and extent of regeneration of peripheral nerves across injury gaps. In this study, we examined basic issues of magnesium metal resorption and biocompatibility by repairing sub-critical size gap injuries (6 mm) in one sciatic nerve of 24 adult male Lewis rats. Separated nerve stumps were connected with poly(caprolactone) nerve conduits, with and without magnesium filaments (0.25 mm diameter, 10 mm length), with two different conduit filler substances (saline and keratin hydrogel). At 6 weeks after implantation, magnesium degradation was examined by micro-computed tomography and histological analyses. Magnesium degradation was significantly greater when the conduits were filled with an acidic keratin hydrogel than with saline (p < 0.05). But magnesium filaments in some animals remained intact for 6 weeks. Using histological and immunocytochemical analyses, good biocompatibility of the magnesium implants was observed at 6 weeks, as shown by good development of regenerating nerve mini-fascicles and only mild inflammation in tissues even after complete degradation of the magnesium. Nerve regeneration was not interrupted by complete magnesium degradation. An initial functional evaluation, determination of size recovery of the gastrocnemius muscle, showed a slight improvement due to magnesium with the saline but not the keratin filler, compared with respective control conduits without magnesium. These results suggest that magnesium filament implants have the potential to improve repair of injured peripheral nerve defects in this rodent model.

Localization and regulation of low affinity nerve growth factor receptor expression in the rat olfactory system during development and regeneration.

Nerve growth factor (NGF), a classic neurotrophic factor, promotes neuronal survival, maintenance, regeneration and differentiation in the peripheral nervous system and parts of the central nervous system. NGF activity is mediated by cell surface bound receptors including the low affinity NGF receptor (LNGFr) which is expressed by some peripheral and central neurons and is present on peripheral nerve Schwann cells during development and regeneration. The olfactory system is a useful model for the study of the role of LNGFr in neuronal development and regeneration. The growth of olfactory axons into the brain begins in the embryo and continues through the first few postnatal weeks. In mature animals there is persistent turnover and generation of olfactory receptor neurons (ORNs) and continuous growth of new axons into the olfactory bulb. These new axons grow along the preexisting olfactory pathway. In the mature olfactory system, LNGFr has been observed in the glomerular layer of the olfactory bulb, the target of ORNs. However, neither the cellular localization nor the developmental expression of LNGFr has been characterized. Here, we tested the hypothesis that LNGFr expression is developmentally regulated in the olfactory nerve and is reinduced following injury to the mature olfactory nerve. LNGFr-immunoreactivity (IR) was first observed in the olfactory mucosa at embryonic day (E)13 and in the olfactory nerve at E14. LNGFr-IR increased in the nerve during embryonic development, began to decrease at around postnatal day (P)5 and was scarcely detectable in normal adults. The staining pattern suggests that LNGFr is located on the olfactory nerve Schwann cells. Streaks of LNGFr-IR were present in the adult olfactory nerve. We reasoned that these streaks might represent transient reexpression of LNGFr associated with normal olfactory neuron turnover and replacement. Consistent with this hypothesis, LNGFr was robustly reexpressed in the adult olfactory nerve following lesion of the olfactory epithelium. Starting late in development (E21) and in the adult, LNGFr-IR was also observed on fibers in deep layers of the olfactory bulb. LNGFr-IR was also observed in neurons of the nucleus of the diagonal band (NDB) in the basal forebrain. NDB is the sole source of cholinergic afferents of the olfactory bulb. Thus, we tested the hypothesis that LNGFr in the deep layers of the olfactory bulb is located on NDB axons by making lesions of NDB. Following the lesion, LNGFr-IR disappeared in the deep layers of the olfactory bulb but remained in the glomerular layer.(ABSTRACT TRUNCATED AT 400 WORDS)

Olfactory receptor neurons express D2 dopamine receptors.

The role of the dopamine (DA) in the olfactory bulb (OB) was explored by determining which of the potential target cells express dopamine receptors (DARs). Previously, it was reported that D2-like DAR (D2, D3, and D4 subtypes) radioligand binding is restricted to the outer layers of the OB. The neuronal elements present only in these layers are the axons of the olfactory receptor neurons (ORNs) and the juxtaglomerular (JG) neurons of the glomerular layer. Based on this pattern of D2-like ligand binding, it was suggested that D2-like receptors might be located presynaptically on ORN terminals. The present study was undertaken to investigate this hypothesis. In the outer bulb layers of rats in which the ORNs were destroyed by nasal lavage with ZnSO(4), D2-like radioligand binding was reduced severely. The receptor subtype D2 mRNA, but not D3 mRNA, was detected in adult rat olfactory epithelial tissue. By using in situ hybridization, this D2 mRNA was located preferentially in epithelial layers that contain ORN perikarya. D2 mRNA was eliminated after bulbectomy, a manipulation known to cause retrograde degeneration of the mature ORNs. Taken together, the surgical manipulations indicate that mature ORNs express D2 DARs and are consistent with the hypothesis that functional receptors are translocated to their axons and terminals in the bulb. This suggests that dopamine released from JG interneurons could be capable of presynaptically influencing neurotransmission from the olfactory nerve terminals to OB target cells through the D2 receptor.

Effects of insulin-like growth factor 1 on olfactory neurogenesis in vivo and in vitro.

The rate of neurogenesis in the peripheral olfactory neuroepithelium is regulated by unknown mechanisms. The members of the insulin-like growth factor (IGF) family can influence neuronal generation, survival and/or differentiation. Several members of this family, in particular IGF-1, are expressed at high levels in the olfactory bulb and epithelium, where they could influence the generation and/or survival of olfactory receptor neurons (ORNs). To explore the role of IGF-1 in the olfactory epithelium (OE), we asked which cells expressed IGF-1 receptors (IGF-1Rs), using olfactory cell cultures and cryostat-cut tissue sections of neonatal (postnatal day four) and adult rat OE. An antibody specific for the alpha subunit of the IGF-1R densely labeled a subset of ORNs but not other cell types in sections and cultures. These ORNs were primarily immature, as determined by double labeling with neuronal markers. The number of IGF-1R-labeled cells as well as the levels of IGF-1R protein (determined by immunoprecipitation and Western blotting) decreased with age, which is consistent with normal developmental changes. To study IGF-1 effects in the intact animal, we infused IGF-1 and related growth factors into the noses of newborn Sprague-Dawley rats, i.e., when the epithelium is still developing. Growth factors or carrier solution (0.9% NaCl with 0.25% bovine serum albumin to prevent nonspecific binding) were applied (10 microliters) to the left nostril once per day starting shortly after birth on postnatal day 1 (P1), P2 and P3, and the animals were sacrificed on P4 by decapitation. After paraformaldehyde immersion fixation, cryostat sections of the olfactory area of the nose were immunostained for the proliferating cell nuclear antigen (PCNA). Sections were position-matched by turbinate structure and then epithelial height and area of PCNA staining at the base of the epithelium (which represents division of primarily neuronal precursors) were measured by image analysis. Both were significantly increased by rat IGF-1 (20 ng/ml, 2.6 nM), but not insulin (20 ng/ml, 2.6 nM) or an IGF-1 derivative, LongR3 IGF-1 (200 ng/ml, 22 nM), that does not bind to the IGF-1 binding proteins (IGFBPs). Thus IGF-1 appears to influence the rate of olfactory neurogenesis, and its actions are not modified by the IGFBPs. These data suggest an important role for IGF-1 in the OE.

Olfactory neurons in vitro show phenotypic orientation in epithelial spheres.

Production and differentiation of olfactory neurons occur in spherical, multi-neuronal aggregates that form in cultures where dissociated newborn rat nasal cells are plated on to CNS glial cells. We show here that neuronal cell bodies were primarily located in the peripheral layers of the spheres, and almost every neuronal sphere contained one or several non-cellular central cavities. The dendrite-like processes of the olfactory neurons, immunostained for neuron-specific tubulin or the olfactory marker protein, were aligned and directed towards the central cavities. Olfactory neurons in the intact animal show a similar relationship with the nasal lumen. Non-neuronal cells formed multiple layers centrally, bordering the cavities. This degree of phenotypic re-creation is unusual in a dissociated monolayer culture system.

2-Mercaptoethanol is a survival factor for olfactory, cortical and hippocampal neurons in short-term dissociated cell culture.

A medium originally designed for lymphocyte growth promoted robust survival of olfactory receptor neurons (ORNs) in short-term (4-day), dissociated cell culture. The key ingredient for survival of neurons in both serum and serum-free conditions was 2-mercaptoethanol (2-ME). Enhancement of survival may be thiol-mediated because two other thiol compounds, 2-mercaptoethylamine and monothioglycerol, also increased ORN survival. Addition of 2-ME also significantly increased survival of embryonic cortical and hippocampal neurons in a serum-free medium, and embryonic cortical neurons in a serum-containing medium. After plating and growth in a serum-free medium containing 2-ME, survival of all three types of neurons was equivalent to, or greater than, survival in serum-containing media. Thus, thiols such as 2-ME promote the survival of multiple types of neurons in short-term cell culture.

Monoclonal antibody to carnosine synthetase identifies a subpopulation of frog olfactory receptor neurons.

A monoclonal antibody directed against the synthetic enzyme for the dipeptide carnosine was used on cryostat sections of olfactory epithelium from the grass frog Rana pipiens. A subpopulation of what morphologically resembled olfactory receptor neurons were immunolabelled by this antibody. Labelled cells were completely stained, including the cell body, axonal and dendritic processes, dendritic knobs and cilia-like projections from the knobs.

Transition between immature radial glia and mature astrocytes studied with a monoclonal antibody to vimentin.

A monoclonal antibody to vimentin (RBA1) and a polyclonal antiserum to glial fibrillary acidic protein (GFAP) were used in double labeling experiments to examine astrocyte intermediate filaments in development and wounding. RBA1 bound to radial glia in newborn rat parietal cortex that are predominantly anti-GFAP-negative. The RBA1-positive radial fibers disappeared by postnatal day 20 with the greatest rate of disappearance occurring between day 8 and day 15. Between birth and day 20, the anti-GFAP staining increased to the adult pattern in mature shaped astrocytes. Some overlay was observed between the binding patterns of the two antibodies. Stab wounds to cortical areas were made at a developmental time when there were normally no RBA1-positive astrocytes. RBA1-positivity was present in some astrocytes but only at the edges of the wounds. The distribution patterns of RBA1-positive cells led to hypotheses concerning the possible function of vimentin in astrocytes and its regulation during development and wounding.

A monoclonal antibody against vimentin: characterization.

A monoclonal antibody was developed using rat cerebral cortex astrocytes purified in vitro as the antigenic material. Screening was done by labeling antibodies bound to cerebellum slices with fluorescent tagged secondary antibodies. The monoclonal antibody (RBA1) bound to intracellular filaments in all cells examined in culture. The coloration of tissue sections by RBA1 was identical to that described for polyclonal antibodies against the intermediate filament protein vimentin. In the brain this included binding to meninges, blood vessels, ependymal cells, choroid plexus lining cells and a subpopulation of astrocytes. The latter included Bergman glial fibers, white matter astrocytes and tanycytes. Müller cells in the retina and fibroblast-like cells in the skin, tongue and intestine were RBA1-positive. In immunoblots in which purified vimentin and desmin were run on SDS and transferred to nitrocellulose paper, RBA1 bound to vimentin but not desmin. When cultured astrocyte proteins were blotted, the antibody bound to both GFAP and vimentin, but no GFAP staining was observed in any of the tissue section staining. Purified vimentin blocked tissue and cultured cell binding of the antibody. Therefore RBA1 is considered to be an antibody specific for the intermediate filament protein vimentin.

Transplantation of septal neurons maintained in long-term culture.

Septal cells were maintained in culture for varying lengths of time and then transplanted to adult hippocampi from which the native septal input had been removed. Cells grafted after 1 week in culture survived transplantation to the brain and innervated the host hippocampus in a pattern comparable to the native cholinergic projection. Cells grafted after 2 weeks in culture did not survive in the brain, but did survive re-plating. In addition, the viability of cells transplanted from culture paralleled the viability of brain tissue taken from donors of equivalent ages, suggesting that changes which occur in culture and affect the viability of transplanted neurons may also occur during normal development.

Characterization of olfactory receptor neurons and other cell types in dissociated rat olfactory cell cultures.

In dissociated cell cultures, control over the cellular environment facilitates study of the differentiation of mature cellular phenotypes. Central to this approach is a rigorous characterization of the cells that reside in culture. Therefore, we have used a battery of cell type-specific antibody markers to identify the cell types present in dissociated cultures of olfactory mucosal cells (containing cells from both the epithelium and lamina propria). To identify olfactory receptor neurons in the cultures, staining with antibodies against neuron-specific tubulin was compared to staining with antibodies to neuron-specific enolase, the neural cell adhesion molecule, N-CAM, and the adhesion molecule, LI. Staining of mature olfactory neurons in culture, with an antibody against the olfactory marker protein, was compared to staining with antibodies to carnosine. In contrast to tissue section staining, the overlap between carnosine and olfactory marker protein staining was not complete. Olfactory nerve glial cells were immunoreactive for the S100 beta protein and nestin, an intermediate filament found in early neuronal progenitor cells and Schwann cells. Antibodies to nestin did not label olfactory neurons or progenitor cells. An antibody to an oligodendrocyte-Schwann cell enzyme, 2',3'-cyclic nucleotide 3'-phosphodiesterase, did not label olfactory glia, but did label oligodendrocyte-like cells that appeared to be derived from the CNS glial feeder layer. An antibody against the heavy (200 kDa) neurofilament protein stained a minor subset of cells. The cultures also contained muscle cells, cartilage cells and macrophages (and/or microglia). These results demonstrate that multiple cell types either maintain or re-establish differentiated, cell type-specific phenotypes in dissociated olfactory cell cultures.

Cultured rat olfactory neurons are excitable and respond to odors.

Newborn rat nasal tissues containing olfactory epithelium were dissociated and maintained in a monolayer cell culture. Neurons were present, as determined by immunostaining with antibodies to 4 neuron-specific proteins: neuron-specific enolase, microtubule-associated protein 2, tau protein and synaptophysin. Immunostained neurons had a distinctive morphology resembling olfactory neurons. By patch-clamp analysis, these cells were electrically active. Responses of some neurons to physiological concentrations of an odorant mixture identified them as olfactory receptor cells.

Age and differentiation-related differences in neuron-specific tubulin immunostaining of olfactory sensory neurons.

Olfactory sensory neurons (OSNs) are unusual mammalian neurons because they are produced continually throughout adult life and because their production is upregulated after injury. Because OSNs also have an unusual immunological profile and do not bind the commonly used antibody markers for neurons, we sought new antibody markers for studies of OSN regeneration. In this report, we characterize the staining patterns of antibodies to the Class III beta, neuron-specific, tubulin (NST) in rat olfactory tissue sections, to determine if these antibodies specifically label OSNs. In tissue sections from newborn rats, monoclonal antibodies to NST labeled cell bodies and processes of both immature (olfactory marker protein, OMP, -negative) and mature (OMP-positive) OSNs. In tissue sections from adult rats, immature OSNs showed both cell body and dendrite staining with anti-NST, while mature OSNs showed little or no cell body staining. Mature OSNs appeared to have both axonal and dendritic anti-NST staining. Axonal staining was suggested by the complete labeling of the olfactory nerve bundles and the nerve fiber layer of the olfactory bulb. The extent of labeling was judged by comparison with anti-OMP staining. Mature OSN dendritic staining was suggested because a much higher number of dendrites were anti-NST stained in the epithelium than cell bodies. These changes suggest both age and differentiation-related changes in subcellular distribution of NST in OSNs. NST antibodies are thus good markers for all OSNs in the newborn rat, but selective markers for immature OSNs and mature OSN processes in the adult rat. NST antibodies may also be useful probes for beta III tubulin function in neurons.

Species differences in amphibian olfactory neuron reactivity to a monoclonal antibody.

A monoclonal antibody immunostained a subpopulation of olfactory sensory neurons in cryostat sections of the olfactory mucosa of the grass frog, Rana pipiens, and the bullfrog, Rana catesbeiana. However, in the olfactory tissues of the African clawed frog, Xenopus laevis, only mucus and mucus-secreting components were stained, and no cell-specific immunoreactivity was seen in the tiger salamander, Ambystoma tigrinum. This antibody is a useful marker of olfactory neuronal subpopulations in some amphibians and illustrates the difficulties in cross-species immunocytochemistry.

Selective gray matter staining of human brain slices: optimized use of cadaver materials.

We report a novel staining technique for human brain slices that distinguishes clearly gray from white matter. Previously described techniques using either Prussian blue (Berlin blue) or phthalocyanine dyes usually have included a hot phenol pretreatment to prevent white matter staining. The technique we describe here does not require hot phenol pretreatment and allows the use of brains stored for postmortem periods of one to two years prior to staining. Our technique involves staining with copper(II) phthalocyanine-tetrasulfonic acid tetrasodium salt 1% in water for 2 h followed by acetic acid treatment; this produces excellent blue staining of gray matter with little white matter staining. The stained brain slices are excellent for teaching human brain anatomy and/or pathology, or for research purposes.

The olfactory nerve contains two populations of glia, identified both in vivo and in vitro.

The peripheral olfactory nervous system exhibits, uniquely, neuronal cell body replacement and reestablishment of central connections in adult mammals. The role of the olfactory nerve glia in these phenomena is unknown, but information might be provided by in vitro systems. This paper reports on the characterization of olfactory nerve glia in dissociated cell cultures of newborn rat nasal mucosal tissues. The predominant type of glial cell resembled Schwann cells and immunostained for the S-100 protein, found in all glial cell types; glial fibrillary acidic protein (GFAP), found in astrocytes and nonmyelinating Schwann cells; and showed binding of 217C, a monoclonal Schwann-cell marker that binds to the low-affinity NGF receptor in glioma cells. They were negative for A2B5. The Schwann-cell-like olfactory glia changed morphology upon culturing in serum-free medium, with further shape changes after plating on laminin. Plating on laminin increased cell numbers. A second population, found only after GFAP-immunostaining, was astrocyte-like in morphology and represented approximately 10 percent of all glial cells. These were S-100-, A2B5-, and 217C-negative, a unique glial cell immunological profile. At low dilutions of anti-GFAP (1/10,000), or with weak fluorescent secondary antibodies, astrocyte-like glia were immunostained but Schwann-cell-like glia were not detectable. Astrocyte-like glia were not an artifact of the dissection, since they were detectable in tissue sections of newborn-rat olfactory nerves immunostained with a low dilution of anti-GFAP. The presence of two types of glial cells in culture suggests similarities between olfactory glia and enteric glia.

Purified cultures of keratin-positive olfactory epithelial cells: identification of a subset as neuronal supporting (sustentacular) cells.

The mammalian olfactory neuroepithelium, lining part of the nasal cavity, retains into adulthood progenitor cells for the olfactory receptor neurons and other cell types in the epithelium. The details of cellular lineage relationships are not completely understood. In particular, the exact nature of the interactions between several progenitor cell types and their relationship to neurons is not known. Studies of this system have been hampered by the lack of cell culture models and insufficient cell-type-specific markers. Antibodies to the cytokeratins are fairly specific markers for one potential progenitor cell type, the dark basal cells of the olfactory epithelium. Keratin immunostaining was used to develop cell culture systems which contained large numbers of putative dark basal cells, using the soft nasal mucosal tissues of both newborn and adult rats. Media and substrate conditions were optimized. The conditions which supported growth of keratin-positive nasal cells for greater than one month, and allowed partial purification, suggested similarities between olfactory and skin keratinocytes. Immunostaining with a monoclonal antibody specific for sustentacular cells (SUS-1) showed a subset of these cells present in culture, with some cells double-labelled with anti-keratin. This staining confirms the olfactory origin of at least a subset of the cells, and supports the proposal that the majority of cells were the dark olfactory basal cells. This culture system gives novel insights into olfactory epithelial cell physiology, and allows culture of these cells for further studies examining regulation of differentiation.

Laminin supports short-term survival of rat septal neurons in low-density, serum-free cultures.

Septal neurons from embryonic rats do not survive for 24 hr when dissociated and cultured at low density in a serum-free medium. Laminin at 5-40 micrograms/ml acts as a survival-promoting agent in the presence of 1 mM pyruvate. Laminin is effective in promoting survival only if it is added before or during cell plating; it does not support survival if added after cell attachment. Variations in laminin concentration do not effect the total number of cells attaching to the culture plate. The percentage of septal cells with neurites is increased in a dose-dependent manner by laminin as early as 45 min after cell plating, suggesting an effect of laminin on the rate of neurite initiation. Neurite extension at 24 hr is also dependent on laminin concentration. This study suggests that some interaction between cells and the substrate, independent of cell attachment, has a profound effect on cell physiology, increasing both cell survival and the rate of neurite extension. This study also defines the survival requirements of septal neurons in a chemically defined environment in a low-density situation where cellular interactions are at a minimum.

In vitro generation of adult rat olfactory sensory neurons and regulation of maturation by coculture with CNS tissues.

Olfactory sensory neurons (OSNs) are continually generated throughout life. Although previous studies have examined neurogenesis in olfactory cell cultures derived from embryonic or newborn rodents, we demonstrate neurogenesis in cell cultures derived from adult rat tissues. Dissociated cells taken from adult rat nasal mucosal tissues (ANM cells) were plated onto a feeder layer of newborn rat cortical glia (astrocytes) in serum-free conditions. Immature OSNs (stained for neuron-specific tubulin, NST) increased in number between 1 and 5 d in vitro (DIV) and in mass thereafter. Mature OSN (stained for olfactory marker protein, OMP) numbers decreased between 1 and 5 DIV, then increased over 5 DIV values by 12 and 15 DIV. Pulse labeling with [3H]thymidine confirmed in vitro neurogenesis. To determine whether the target cells for OSNs, olfactory bulb (OB) neurons, provide trophic support, dissociated newborn rat OB cells were cocultured with ANM cells on glia. This resulted in greater numbers of OMP-positive (OMP+) neurons after 9 DIV than ANM-alone cultures. This neurotrophic effect was not OB specific. Addition of newborn rat cerebellar and embryonic rat ventral mesencephalic cells to ANM cells also increased OMP+ neurons, whereas addition of newborn rat cortical cells or controls (purified glia or fibroblasts) did not. Changes in numbers of dopaminergic neurons (stained for tyrosine hydroxylase), present in OB and VM cultures, did not correlate with OMP+ neuronal increases. Thus, cultures of adult rat OSNs demonstrate neurogenesis, and trophic/maturation support is variably provided by CNS neurons (and not glia).