Albert I. Farbman

Olfactory marker protein during ontogeny: Immunohistochemical localization☆

Abstract Specific immunohistochemical staining for the olfactory marker protein (OMP) is first demonstrated in rat olfactory receptor neurons at embryonic day 18, at which age no OMP can be seen in the olfactory bulb or vomeronasal epithelium. At 21 days OMP staining in the olfactory epithelium is more extensive and is evident in the fibrous and glomerular layers of the bulb as well. Staining intensity increases progressively until the full adult pattern is seen by 1 month postnatally. In the vomeronasal organ, staining is not observed until the fourth postnatal day and, even then, only with higher antiserum concentrations. In mice, very similar results are obtained, except for a much earlier appearance of OMP, on embryonic day 14. Olfactory epithelium from 12- and 13-day rat embryos maintained in organ culture for up to 2 weeks did not exhibit OMP staining, nor did several neural or nonneural tissues from adult animals. The temporal and causal interrelationships between OMP and other indicators of olfactory receptor cell maturation are considered.

Olfactory neurogenesis: genetic or environmental controls?

Vertebrate olfactory neurons are unique among neurons in that they are continually replaced throughout the life of the animal. The rate of neurogenesis can be regulated by manipulating the system to abbreviate or prolong the average life of a sensory neuron. Moreover, the neuron may die before or after reaching full maturity. When compared with other neurons, the fully mature olfactory neuron retains juvenile characteristics; it is probable that genetic controls operate to maintain this relatively immature state.

Ablation of the olfactory bulb up-regulates the rate of neurogenesis and induces precocious cell death in olfactory epithelium

Young adult rats were unilaterally bulbectomized and tritiated thymidine ([3H]TdR) was injected at variable times following surgery to determine the effect of bulbectomy on the rates of cell proliferation and cell death in the olfactory epithelium. Removal of the olfactory bulb elicits a two- to fourfold increase in the proliferation rate of ipsilateral olfactory epithelial cells 7-50 days following surgery. On the contralateral side, there was a temporary twofold increase in the proliferation rate during the second week after surgery, but this returned to control values at 3 weeks. This temporary increase was in parallel with the response on the ipsilateral side so that the ratio between operated and unoperated sides remained at two. Cell death in olfactory epithelium is also up-regulated following bulbectomy. Death of cells can occur as early as 1 day following incorporation of [3H]TdR, i.e., well before the sensory neurons become mature. This means there is an over-production of sensory cells, and they die at all stages of their life cycle. The number of cells dying is greater after bulbectomy, indicating that the overproduction of olfactory cells is more pronounced after surgery.

Development of olfactory receptor neuron selectivity in the rat fetus

Olfactory receptor neurons begin to differentiate from stem cells on day E10 of embryonic life in the rat. By day E16, the receptor epithelium is well populated. On this day single neuron action potentials could be recorded with some ease and the electro-olfactogram was well developed. The receptor neurons were functional in that they responded to the vapors of odorous substances. However, they were not selective. Each cell responded to nearly all of the substances in the stimulus set. The first synaptic connections between receptors and mitral cells are established on day E18. The olfactory marker protein is reported to appear first in the receptors on the same day. By day E21, single unit responses changed dramatically. The cells became selective, responding to about half of the substances in our set. The electro-olfactogram reached its limiting amplitude well before this time.

Early development of olfactory receptor cell axons

An electron microscope study was done on development of olfactory receptor cell axons in rat fetuses 13-17 days after conception (E13-E17). Initiation of axon outgrowth was first seen on E13. On E14, small bundles of olfactory axons, accompanied by epithelial cells, grow out of the epithelium and, by so doing, breach the basal lamina. Close examination of these epithelial cells from E14 fetuses has revealed that they can be grouped into two types, one containing a ribosome-rich, dense cytoplasm, the other containing fewer free ribosomes and a more lucent cytoplasm. The first of these types is present infrequently or not at all at later times in development. The fate of the migrating cells is not known. Another notable observation was the indentation of olfactory epithelium by blood vessels during the developmental stages studied. There is very close association between processes of vascular endothelium and cells of the olfactory epithelium, so close, in fact, that a basal lamina is frequently absent. It is possible that vascular endothelium plays a role in breakdown of basal lamina, thus enabling olfactory axons to breach this barrier as they leave the epithelium.

Electron microscope study of palate fusion in mouse embryos.

Abstract An electron microscope study of palatal fusion in 14 1 2 - day mouse embryos was done. The epithelium of the palatine processes was examined prior to, during, and immediately following fusion. Significant findings include the following. In the prefusion palatine process, the basement lamina underlying the epithelium was discontinuous and there were epithelial cell projections into the connective tissue space. At the time of fusion no membrane specializations were found on contacting surface cells of opposing palatal processes. There was evidence, however, of true adhesion between epithelia of opposing processes, but no extracellular “sticky substance” was demonstrable. Immediately following palatal fusion there was widespread evidence of cell death in the midline epithelial seam. Dead epithelial cells were phagocytized by neighboring epithelial cells in the fusion line and by invading connective tissue cells.

Olfactory afferent regulation of the dopamine phenotype in the fetal rat olfactory system

Recent studies strongly suggest that functional olfactory receptor cell innervation is necessary for the maintenance of the dopamine phenotype in the adult rat olfactory bulb. To determine whether afferent innervation is required for the initial expression of the dopaminergic phenotype during development, the current studies investigated the association between afferent innervation and phenotypic expression using both in vivo and in vitro systems. Ontogeny of the dopamine phenotype in the rat main olfactory bulb was assessed by the appearance of immunoreactivity for tyrosine hydroxylase, the first enzyme in the dopamine biosynthetic pathway. Development of receptor afferent innervation of the bulb was demonstrated with olfactory marker protein immunoreactivity. Tyrosine hydroxylase-immunoreactive cells occurred only in regions of the olfactory bulb receiving afferent innervation. However, the appearance of afferent fibers in the olfactory bulb preceded tyrosine hydroxylase expression by three to four days (gestational days 14-15 versus 18, respectively). In explant cultures, significant numbers of tyrosine hydroxylase-containing cells were observed only in en bloc co-cultures of presumptive olfactory bulb and epithelium. Explant cultures of presumptive olfactory bulb alone contained few, if any, tyrosine hydroxylase-immunoreactive cells. Similarly, explants produced by recombining previously separated presumptive olfactory bulb and epithelium exhibited very few tyrosine hydroxylase-immunostained cells. These data suggest that expression of the dopamine phenotype, as indicated by the presence of tyrosine hydroxylase, depends on a critical level of afferent innervation. The results also support previous studies which indicated that neuronal activity or an activity-dependent process may be required for induction of tyrosine hydroxylase expression.

Transforming growth factor-α and other growth factors stimulate cell division in olfactory epithelium in vitro

The rate of cell division in olfactory epithelium (OE) is upregulated by ablation of the olfactory bulb (Carr and Farbman, 1992), or downregulated by occlusion of a naris. We used an organ culture assay of fetal rat olfactory mucosa to study regulation of the mitotic rate. Addition of any one of three members of the epidermal growth factor (EGF) family-EGF, transforming growth factor-alpha (TGF-alpha), or amphiregulin (AR)-to a serum-free culture medium resulted in a two- to threefold increase in the number of dividing OE cells. TGF-alpha elicited a maximal response in a dose of 100-200 pM culture medium and was 2 orders of magnitude more potent than the other EGF family members. Addition of TGF-beta 1, TGF-beta 2, insulinlike growth factor-1 or platelet-derived growth factor to the culture medium had slightly less effect than EGF or AR, in about the same molar dose range; addition of nerve growth factor had virtually no net effect on cell division. Immunohistochemistry on adult rat OE showed that basal cells, supporting cells, and acinar cells of Bowmans glands were immunoreactive with antibody to TGF-alpha but not with antibody to EGF. Most growth factors upregulated division of both olfactory neuron progenitors and supporting cells. The data suggest that several growth factors, most prominently TGF-alpha 1, may participate in the mitotic regulation of OE.

Supporting cells as phagocytes in the olfactory epithelium after bulbectomy

Macrophages are known to be phagocytes in the olfactory epithelium of adult rats. The participation of other cell types in phagocytosis in association with the cell death process was examined in the olfactory epithelium after unilateral bulbectomy of neonatal mice. The terminal deoxynucleotidyl transferase (TdT)‐mediated biotinylated dUTP nick end‐labeling (TUNEL) method revealed that the process of olfactory cell death consists of acute and chronic periods. The number of apoptotic cell profiles on the operated side peaked at 1 day, and the percentage of labeled cell profiles was 13.6%. The number of dying cells rapidly decreased at 3 days and decreased further at 5 days. Only 3% of the cells were labeled at 5 days. The percentage of dying cells increased again at the end of first postoperative week and remained two‐ to four‐fold higher than control values for 2 months (4.7–5.3%). Electron micrographs of sections from early postbulbectomy stages (1–7 days) showed that as many as 30% of supporting cell profiles contained apoptotic bodies, cellular debris, and phagosomes in the cytoplasm. The number of supporting cell profiles containing phagosomes declined to a plateau 2 weeks following bulbectomy and remained at 8–12% of the supporting cell population for 2 months. The results indicate that supporting cells in the olfactory epithelium play a significant role in phagocytosis in both acute and chronic periods of cell death after bulbectomy in newborn mice. However, supporting cells are not the exclusive phagocytic cell type in the bulbectomized epithelium; a small number of macrophages was also observed. Moreover, the phagocytosis by supporting cells was observed in unperturbed epithelium in the early stages during postnatal development.

Proliferation in the vomeronasal organ of the rat during postnatal development.

We investigated proliferation of sensory cell precursors in the rat vomeronasal organ (VNO) at various postnatal ages from birth (P1) to P666. In the rat, which continues to grow during most of its adult life, proliferation might be related to growth and/or replacement. Proliferating cells were labelled by BrdU injection, and histological sections of the VNO were evaluated after immunohistochemical detection of BrdU. Proliferation density (number of proliferating cells/section) decreased dramatically from 115 at P1 to 27.2 at P21, although the area increased. Adult values were reached at P66–P333 (10.3 cells/section); at P400–P666 the value was 8.6 cells/section. Distribution of labelled cells changed considerably with age: in neonates the cells were nearly equally distributed throughout the sensory epithelium, whereas from P21 onwards most proliferating cells were concentrated in clusters near the boundaries with non‐sensory epithelium. Labelled cells in the sensory neuronal layer were adjacent to the undulating basement membrane‐bordering capillaries that intrude into the sensory epithelium, indicating that they were true basal cells. The volume of the sensory epithelium increased between P1 and P66, and remained constant thereafter, although the length still increased. Length and volume of the sensory epithelium were related to body size, not to sex; males and females of the same body size had the same VNO size. The complex changes in proliferation pattern during postnatal development indicate differential growth and replacement. We suggest that in adults the labelled cell clusters near the boundaries are a pool for growth, whereas proliferation in the central parts represents a replacement pool.