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It has been shown in previous studies that the marsupial central nervous system is born at a relatively immature state. Although olfaction is thought to play a role in guiding the locomotion of the newborn, the cellular substrates on which this notion is based have not been systemically investigated. This review article summarises the anatomical development of the primary olfactory pathway in the postnatal Monodelphis. The olfactory epithelium and bulb appear morphologically immature at birth although some of the olfactory neurons are shown to express olfactory marker protein. The olfactory tissues subsequently undergo a rapid sequence of developmental events during the first two postnatal weeks. The evidence shows that the marsupial and eutherian olfactory system share a similar temporal sequence of developmental processes although the former proceeds at a lag time of about 10-14 days compared to that a mice (using the date of birth as a common reference point). Much physiological and behavioral studies remain to be done before we can be certain about the time at which functional maturity is attained in this system.

We have examined the expression of the MT1 and MT2 isoforms of metallothionein in the mouse olfactory mucosa. In untreated mice, metallothionein was strongly expressed in supporting cells, acinar cells of the Bowman's glands, and olfactory neurons. Expression was however restricted to a subset of cells within each type, and to zones within the olfactory system. Irrigation with ZnSO4 solution caused exfoliation of the olfactory epithelium and during the resultant regeneration, metallothionein immunoreactivity was associated with the proliferating basal cells. The ability to express MTs 1 and 2 did not appear to be obligatory for the early stages of regeneration since mice which do not express these isoforms responded similarly to wild type mice. Strong nuclear expression of metallothionein was noted in the untreated olfactory chamber following unilateral irrigation.

Ultrastructural observations in the grey short-tailed opossum (Monodelphis domestica) and rat revealed that ensheathing cells were intimately related to the early formation of olfactory axons. Whilst the axons were still in the olfactory epithelium, they were enveloped by ensheathing cell processes which formed a cradle-like structure on the basal side of the epithelium. Continued downgrowth of the axons towards the lamina propria resulted in an evagination with the ensheathing cell process or cell body in direct contact with the basal lamina. Subsequently the basal lamina became fragmented, and the newly formed olfactory nerve emerged from the olfactory epithelium. As the olfactory nerve grew, it was observed that the ensheathing cell process always extended ahead of the axons while axon terminals moving ahead of ensheathing cells were never observed. The findings in this study suggest that ensheathing cells play a role in regulating and promoting olfactory axon growth.

The response of olfactory Schwann cells was assessed at 2, 4, and 7 days following intranasal zinc sulfate irrigation in 1-month-old mice. Ultrastructural and immunohistochemical observations showed dramatic differences between experimental and control mice which had been washed with saline intranasally. Two days after zinc sulfate treatment, many olfactory nerve bundles contained patchy areas of axonal degeneration, while the cell bodies of the olfactory Schwann cells appeared to have increased in electron density and to have shifted peripherally. Some of the cell bodies protruded from the surface of the axon fascicle, suggesting that the olfactory Schwann cells were in the initial process of migrating away. On the fourth day when most of the olfactory axons had degenerated, some olfactory Schwann cells were aligned immediately beneath the basal lamina of the olfactory epithelium. These cells were immunopositive for the S-100 protein and possessed an expanded perinuclear space. Many olfactory Schwann cells were present in the region beneath the cribriform plate, while some appeared to have passed through the gaps between the bony plates to reach the olfactory bulb. Hence, the results showed that many olfactory Schwann cells migrated towards the olfactory bulb following loss of axonal contact. Furthermore, on the seventh day following zinc sulfate treatment, some olfactory Schwann cells in the vicinity of the olfactory bulb appeared phagocytic, as indicated by their extension of processes around fragments of cell debris and the presence of lysosome-like organelles in the perikaryon. The control mice which had been intranasally irrigated with saline did not demonstrate massive olfactory axonal degeneration, and the morphology of the nasal cavity region was similar to that of normal mice.

Morphological and biochemical methods were applied to assess the effects of implanting cultured astrocytes into the hemisected adult rat spinal cord. Astrocytes were purified from neonatal rat cortex and introduced into the lesioned spinal cord either in suspension injection or cultured on gelfoam first. The control groups were rats which had hemisection with injection of culture media or with gelfoam grafted alone. At various time points after surgery (two weeks to two months), the spinal cord was removed and processed for routine light microscopy, immunofluorescence, gel electrophoresis and immunoblotting. As early as two weeks after surgery, a significantly smaller volume of scar tissue was consistently found in the experimental groups. This reduced scarring was also confirmed by immunofluorescence staining and immunoblotting for glial fibrillary acidic protein in the specimens two months after hemisection. Compared to the control groups, the experimental groups also had more intense staining for neurofilaments, which was confirmed by immunoblotting. However, labelling of the astrocytes with Phaseolus vulgaris leucoagglutinin conjugated with fluorescein showed that the astrocytes migrated at a rate of 0.6 mm/day from the original implanted site. The results therefore suggested that the cultured astrocytes probably exerted their effects over a short time period (less than two weeks) around the lesion site. They could have altered the microenvironment and as a result less scar tissue was formed. Hence, there was less barrier to the regrowth of nerve fibres.

The morphological and biochemical changes were studied in adult Sprague-Dawley rats after hemisection at the L3 spinal cord level. After survival periods of one, two and three months, fluorescent tracers, FluoroGold or rhodamine B, were implanted into the dorsal white columns of these rats at the positions of the corticospinal tract below the lesion. Following uptake of the tracer, the rats were killed and the motor cortices and spinal cords of both control and hemisected rats were analysed for positively labelled neurons. The highest number of labelled cells were found two months after hemisection. They were present in both sides of the cortices, particularly in the contralateral cortex, and also in the gray matter of the spinal cord above the hemisection. A few rats which were subjected to complete transection of the spinal cord also showed labelling of neurons in the motor cortex two months after lesion. The Protargol silver technique and the [3H]choline uptake study confirmed the presence of nerve fibres traversing the lesion site in the hemisected spinal cord. Furthermore, when the rats that had been hemisected two months earlier were subjected to a second cut at the same site, chromatolytic neurons were observed in the spinal cord as well as in the motor cortices of both sides. The hemisected rats demonstrated limited recovery in limb movement. The evidence of this study clearly shows that sprouting of nerve fibres has occurred in the lesioned adult rat spinal cord.

Olfactory neurons dissociated from the olfactory mucosa of 4-5-week-old Sprague-Dawley rats are plated on either monolayers of ensheathing cells or cortical astrocytes. It is found that the ensheathing cells support a slightly higher percentage of neurite-bearing olfactory neurons than the astrocytes. Scanning electron microscopy shows that some of the cytoplasmic extensions of the ensheathing cells are closely associated with the olfactory axons while others appear to ensheath them. Olfactory neurons grown on uncoated, poly-L-lysine or laminin-coated glass coverslips in the presence of medium conditioned by ensheathing cells fail to grow neurites, suggesting that interaction between membrane molecules, and not trophic factors, may be required for neurite growth. However, it is unlikely that these membrane molecules are L1 and N-cadherin because immunohistochemical staining shows that only a small proportion of the cultured ensheathing cells express L1 (9%) and N-cadherin (24%).

We have derived highly enriched populations of ensheathing cells (ECs) from the olfactory nerve layer of neonatal rat olfactory bulbs. Contaminating cells, such as fibroblasts, were eliminated from EC cultures by cytosine arabinoside and immunoadsorption with antiserum to Thy-1.1. At the same time, ECs were stimulated to divide by the addition of bovine pituitary extract into the culture media. Confluent cultures containing 96-99% ECs, were comprised of either spindly bipolar cells or cells bearing multiple processes oriented on opposite poles. The ECs immunostained positively for GFAP, S-100 protein, N-CAMs and Neu 5, and were negative for the presence of neurofilaments. Scanning and transmission electron microscopy showed that the ultrastructure of the ECs resembled that in vivo. The nucleus was irregular in shape, intermediate filaments were usually scattered throughout the cytoplasm instead of being grouped into bundles, and rough endoplasmic reticulum existed as isolated expanded profiles.

Despite increasing knowledge about the biophysiology of the human olfactory system, understanding of the development of this pathway in humans lags considerably behind that of other vertebrates. Developmental studies have largely concentrated on the generation of cell types in the olfactory epithelium during the first trimester, while detailed ultrastructural observations usually describe the adult morphology. In this review, we have shown that contrary to what has been generally assumed, the surface of the human olfactory epithelium is heterogeneous and that its olfactory nerves differ ultrastructurally from those of other vertebrates studied. The development of the human primary olfactory pathway is discussed in terms of the appearance of olfactory bulb laminae, synaptogenesis and the expression of specific cell markers, such as the S-100 protein and olfactory marker protein (OMP). Positive immunohistochemical staining for N-cadherin in human fetuses suggests that growth of olfactory axons to their target may be mediated by cell adhesion molecules. The overall data presented here indicate that this pathway develops more precociously in humans than in rodents. Whether this translates also to earlier functional maturity remains to be elucidated.

Olfactory tissues from human fetuses (17.5-28 weeks of gestation) were stained by immunofluorescence for neural cell adhesion molecules (N-CAMs). Staining for N-CAMs was most prominent in the olfactory nerve bundles in the lamina propria, while in the olfactory epithelium, it was present on the olfactory receptor neurons and globose basal cells. The basal cells proper and supporting cells lacked N-CAMs. In the olfactory bulb, only the olfactory nerve and glomerular layers showed moderate labeling for N-CAMs. Western blot analysis showed that the N-CAMs of the fetal human primary olfactory pathway consisted of three molecular isoforms, N-CAM180, N-CAM140 and N-CAM120.

Olfactory epithelial neurons in mammals are unique in that they continue to differentiate from precursor cells in the adult. These neurons extend long axons into the olfactory bulb. Previous attempts to grow these cells in dissociated cell cultures at low density, have not been entirely satisfactory. We report that when plated at very low density, rat olfactory epithelial neurons will differentiate morphologically and biochemically when cultured on astrocytes, but not on non-cellular substrata, such as polylysine, laminin or fibronectin. We demonstrate with antibodies, that these olfactory epithelial neurons require N-CAM, N-cadherin and L1 for neurite extension. Furthermore, synthetic cadherin-peptides containing the tripeptide HAV which is found in the first extracellular domain of N-cadherin, as well as the amino acids flanking this region, appear to be important in cadherin-mediated neurite growth on astrocytes. Astrocytes also appear to enhance the survival and differentiation of olfactory epithelial neurons from embryonic day 15 and 4-5 week post-natal rats, but this effect is not sustained beyond 5 days in cultures of postnatal epithelium.

In this morphological and immunohistochemical study we show that olfactory schwann cells (OSC) are derived from precursor cells residing in the olfactory epithelium. During development, they migrate out of the epithelium and extend processes to ensheath the olfactory axons. Olfactory mucosa from E14 rat embryos and juvenile rats were treated with trypsin-pancreatin to remove the underlying connective tissue. The epithelial explant was then maintained for two days in culture, during which cells migrated out from the explant. Among them were spindly bipolar cells which were identified as OSC by their positive immunoreaction for glial fibrillary acidic protein, ultrastructure, and association with growing axons. Axonal growth was significantly more profuse in the embryonic explants, in which the polarity of the OSC was oriented parallel with the axons. Ultrastructural observations showed that ensheathment of the bundles of axons resembled those in vivo.

Amitriptyline (AT) was injected i.p. (0.01 g/kg) into newborn rats for 12 days. Stereometric measurements of coronal sections of the olfactory bulbs showed that the area of the external plexiform layer (EPL) in the AT-treated group was significantly smaller than that of control rats. Rats treated with AT also showed a significantly lower increase in body weight compared with the saline-injected control group.

The anterior part of the mammalian nasal septum (NS) persists throughout the life span as hyaline cartilage, in contrast to cartilage in most parts of the body, which is gradually replaced by bone during development. In this study, we have cultured differentiating rat NS under various experimental conditions in an attempt to gain some insight into the osteogenic potential, if any, of the NS and its surrounding connective tissue. Differentiating NS from E15 and E19 rat embryos were dissected and grown under the following conditions: 1) organ cultured in Waymouth's medium or modified Eagle's medium, with or without serum; 2) cultured on chick chorioallantoic membrane (CAM); 3) implanted under rat kidney capsule (KC). Bone-like substance (BLS) never developed in organ cultures, but was observed in CAM cultures and KC implants after 7 days. The BLS was located external to the perichondrium of the NS and was stained red by the van Gieson's technique, indicating the presence of mature collagen. Further evidence of its bone-like characteristics was demonstrated by the presence of alkaline phosphatase and type I collagen. The CAM and KC represent two experimental conditions under which progenitor cells in the nasal septum area may be induced to synthesize BLS.

The maturation of olfactory receptor cells is facilitated by the presence of their target tissue, the olfactory bulb. Organ cultures of embryonic rat olfactory mucosa (OM) maintained in the presence of the presumptive olfactory bulb (POB) had a significantly higher (1.6 X) density of ciliated dendritic knobs than those without the POB. No significant difference was found in the density of non-ciliated dendritic knobs and total knob density in these two groups of cultures. A control group of explants in which the OM and POB had been separated and recombined also showed an increased ciliated dendritic knob density. The area of the olfactory epithelium was the same whether or not the POB was present. In addition, scanning electron microscopy observations revealed a high degree of heterogeneity in the surface morphology of the olfactory epithelium.

The tricyclic antidepressant drug amitriptyline has a detrimental effect on neurite outgrowth in primary explant cultures containing either olfactory receptor neurons or cerebral neurons, from both rat and chick embryos. When the drug is added to the culture medium in doses similar to the plasma concentrations known to be therapeutic in humans, the number of explant cultures expressing neurites is significantly reduced. In higher doses, amitriptyline reduces the amount of olfactory marker protein synthesized by organ cultures of olfactory mucosa.

Light microscopic observations indicated that cellular differentiation of the primary olfactory pathway in human fetuses was relatively advanced by the end of the first trimester. However, immunohistochemical staining showed that olfactory marker protein was present in the receptor cells only at about 28 weeks post-conception. At about 32 weeks, modest amounts of the protein could be found in the peripheral olfactory nerve layer and a few glomeruli, with increased staining observed in subsequent weeks. The results show conclusively that human fetal olfactory neurons synthesize olfactory marker protein during the later half of gestation.

The ultrastructure of differentiating rat presumptive olfactory bulb in organ culture was investigated with particular reference to mitral cell differentiation and formation of synapses. The presumptive olfactory bulb and olfactory mucosa were dissected en bloc from rat embryos on the fifteenth day of gestation and cultured for 7 days, after which the explants were examined by electron microscopy. The presumptive olfactory bulb had differentiated into a laminated structure with layers corresponding to the glomerular, external plexiform and mitral cell layers. Mitral-like cells were identified by their location and large cell size. Ultrastructural observations indicated that they were relatively well-differentiated. Their dendrites extended into the glomerular layer in which they were postsynaptic to incoming olfactory axons. The distal part of these dendrites frequently contained coated vesicles. Both asymmetrical and symmetrical synapses were found. The symmetrical synapses involved dendrodendritic contacts between periglomerular cells. Synapses in reciprocal arrangements were not observed in the organ cultures.

In vitro studies on E15 rat embryos have shown that the number of receptor neurons containing olfactory marker protein (OMP) is markedly increased when olfactory mucosa (OM) is cultured in direct contact with the presumptive olfactory bulb (POB). This facilitatory influence is tissue-specific; that is, it is absent when the POB is substituted with other nervous or non-nervous tissues. In the present quantitative immunohistochemical study, we show that the enhancing influence of the POB is also a stage-specific phenomenon. The number of OMP-containing receptor cells is greatest when E15 OM is cultured with POB of the same age. If the POB was taken from a less mature (E13) or from a more mature (E17) embryo, the number of OMP-containing cells was greatly reduced.