Ultrastructural localization of G-proteins and the channel protein TRP2 to microvilli of rat vomeronasal receptor cells.

Microvilli of vomeronasal organ (VNO) sensory epithelium receptor cells project into the VNO lumen. This lumen is continuous with the outside environment. Therefore, the microvilli are believed to be the subcellular sites of VNO receptor cells that interact with incoming VNO-targeted odors, including pheromones. Candidate molecules, which are implicated in VNO signaling cascades, are shown to be present in VNO receptor cells. However, ultrastructural evidence that such molecules are localized within the microvilli is sparse. The present study provides firm evidence that immunoreactivity for several candidate VNO signaling molecules, notably the G-protein subunits G(ialpha2) and G(oalpha), and the transient receptor potential channel 2 (TRP2), is localized prominently and selectively in VNO receptor cell microvilli. Although G(ialpha2) and G(oalpha) are localized separately in the microvilli of two cell types that are otherwise indistinguishable in their apical and microvillar morphology, the microvilli of both cell types are TRP2(+). VNO topographical distinctions were also apparent. Centrally within the VNO sensory epithelium, the numbers of receptor cells with G(ialpha2)(+) and G(oalpha)(+) microvilli were equal. However, near the sensory/non-sensory border, cells with G(ialpha2)(+) microvilli predominated. Scattered ciliated cells in this transition zone resembled neither VNO nor main olfactory organ (MO) receptor cells and may represent the same ciliated cells as those found in the non-sensory part of the VNO. Thus, this study shows that, analogous to the cilia of MO receptor cells, microvilli of VNO receptor cells are enriched selectively in proteins involved putatively in signal transduction. This provides important support for the role of these molecules in VNO signaling.

Odorants as cell-type specific activators of a heat shock response in the rat olfactory mucosa.

Heat shock, or stress, proteins (HSPs) are induced in response to conditions that cause protein denaturation. Activation of cellular stress responses as a protective and survival mechanism is often associated with chemical exposure. One interface between the body and the external environment and chemical or biological agents therein is the olfactory epithelium (OE). To determine whether environmental odorants affect OE HSP expression, rats were exposed to a variety of odorants added to the cage bedding. Odorant exposure led to transient, selective induction of HSP70, HSC70, HSP25, and ubiquitin immunoreactivities (IRs) in supporting cells and subepithelial Bowman's gland acinar cells, two OE non-neuronal cell populations involved with inhalant biotransformation, detoxification, and maintenance of overall OE integrity. Responses exhibited odor specificity and dose dependency. HSP70 and HSC70 IRs occurred throughout the apical region of supporting cells; ubiquitin IR was confined to a supranuclear cone-shaped region. Electron microscopic examination confirmed these observations and, additionally, revealed odor-induced formation of dense vesicular arrays in the cone-like regions. HSP25 IR occurred throughout the entire supporting cell cytoplasm. In contrast to classical stress responses, in which the entire array of stress proteins is induced, no increases in HSP40 and HSP90 IRs were observed. Extended exposure to higher odorant doses caused prolonged activation of the same HSP subset in the non-neuronal cells and severe morphological damage in both supporting cells and olfactory receptor neurons (ORNs), suggesting that non-neuronal cytoprotective stress response mechanisms had been overwhelmed and could no longer adequately maintain OE integrity. Significantly, ORNs showed no stress responses in any of our studies. These findings suggest a novel role for these HSPs in olfaction and, in turn, possible involvement in other normal neurophysiological processes.

Development and further characterization of a small subclass of rat olfactory receptor neurons that shows immunoreactivity for the HSP70 heat shock protein.

We previously described a rat olfactory receptor neuron (ORN) subpopulation [the 2A4(+) ORNs] that shows uniquely strong reactivity with antibodies to the 70-kD heat shock protein (HSP70) family of molecular chaperones (Carr et al. [1994] J. Comp. Neurol. 348:150-160). The 2A4(+)ORNs are dispersed through zones II-IV of the olfactory epithelium (OE), and their axons project to only two or three glomeruli that are located consistently in each olfactory bulb (OB). To date, the 2A4(+)ORN subpopulation is the only cell population to show such distinct HSP70 immunoreactivity as well as the most discrete ORN subpopulation to be so labeled. The present report shows that 2A4(+)ORN neurons first appear between postnatal days 7 (P7) and P10. Initially, low cell numbers rise to a density of 0.1 2A4(+)ORNs/mm OE length by P14, plateau at 0.9 2A4(+)ORNs/mm by P49, then fall to adult values of 0.4 cells/mm. Autoradiographic birthdating indicates that almost all of these early appearing 2A4(+)ORNs are generated postnatally, in contrast to the prenatal generation of all ORN subpopulations characterized to date by their expression of olfactory receptor protein mRNAs. A developmentally related increase in the mean depth of 2A4(+)ORNs within the OE also occurs. In the OB, initial 2A4(+)axonal projections are to only two or three glomeruli, as in adults. Slight but significant rostral shifts in (+)glomerular location occur with development. The 2A4(+)ORN immunoreactivity was found to be due to expression of HSP70, the dominant stress-inducible member of the HSP70 family, rather than constitutively expressed HSC70. In addition, despite their presence in rat OE, no 2A4(+)ORNs were found in mice, gerbils, guinea pigs, or hamsters.

An enhanced olfactory marker protein immunoreactivity in individual olfactory receptor neurons following olfactory bulbectomy may be related to increased neurogenesis.

Olfactory marker protein (OMP) is a 19-kD acidic protein found throughout the cytoplasm of mature olfactory receptor neurons (ORNs). Its function remains unknown. Following olfactory bulbectomy, the proportion of ORNs mature enough to express OMP declines greatly. However, in the few remaining mature ORNs, it has been observed that the intensity of OMP immunoreactivity (IR) appears to increase over that of ORNs on the unoperated side. We have now investigated this phenomenon quantitatively in rats subjected to unilateral olfactory bulbectomy. Results show that at all postbulbectomy survival periods examined quantitatively (3 days to 6 months), a significant decrease (19-37%) occurs in the transmission of incident light through OMP(+)-ORNs in bulbectomized versus unoperated olfactory epithelium (OE). Further, we also observed a consistent side-to-side difference in OMP IR in control unoperated animals. Possible explanations for these observations and their relation to the still unknown function of OMP are discussed. To test the possibility that OMP might serve a mitogenic role in the OE, recombinant OMP was added to organotypic explant cultures of fetal olfactory mucosa. Addition of OMP resulted in a dose-dependent increase in the density of bromodeoxyuridine-positive cells in the cultures, with a 50% increase occurring at the plateau OMP concentration of 25 pM.

Initial development of a small subclass of rat olfactory receptor neurons characterized by antigenicity to HSP 70.

We have described a subclass of rat olfactory receptor neurons (ORNs) that constitutively shows immunoreactivity with a monoclonal antibody (2A4) directed to the 70-kDa heat shock protein. These ORNs are scattered nonuniformly in olfactory epithelium (OE) Zones II-IV and project to just 2-3 glomeruli at consistent locations in the ventrolateral and ventromedial olfactory bulb (OB) via consistent pathways. To examine early neurogenesis of this subpopulation, paraffin sections from embryonic day 14 (E14) to postnatal day 63 (P63) rats were examined using immunoperoxidase techniques. Results show: (i) a few faintly reactive 2A4(+) ORNs first appear between P7 and P10. Their numbers (and immunoreactivity (IR) intensity) increase to adult levels by P21, reach a peak density approximately twice that of adults by P49, and then decline to adult values by P56. (ii) tritiated thymidine [3H]TdR autoradiographic birthdating studies show that the vast majority of 2A4(+) ORNs present at P21, when adult 2A4(+) ORN densities are first observed, were 'born' postnatally, between P3 and P10. (iii) The initial 2A4(+) ORN OE zonal distribution is the same as in adults. (iv) Through P21 2A4(+) ORN cell bodies are situated quite apically within the OE, but then assume more basal locations as well. (v) In the OB, glomeruli showing 2A4(+) axons appear in some animals as early as P14 and in all animals by P21. Initial location of the (+) glomeruli is similar to that of adults, despite extensive growth and development postnatally. The postnatal neurogenesis of the 2A4(+) ORNs, in contrast to the very early (E13) initial appearance of ORN subclasses characterized on the basis of their putative olfactory receptor mRNAs, indicates that different ORN subclasses may vary in the time of their initial neurogenesis.

Small subclass of rat olfactory neurons with specific bulbar projections is reactive with monoclonal antibodies to the HSP70 heat shock protein.

As part of a study of turnover of rat olfactory receptor neurons we have been examining immunohistochemical expression of members of the 70 kD heat shock protein (HSP70) family in the olfactory epithelium. Expression of HSP70 family members is up-regulated in many cells following exposure to physiologically stressing conditions. Because dying neurons are likely to undergo some sort of physiological stress before the onset of frank degeneration, we hoped that anti-HSP70 monoclonal antibodies would prove to be useful markers for early stages of olfactory neuron cell death. Two anti-human HSP70 monoclonal antibodies were used, Mabs 2A4 and 3a3. Two-dimensional gel electrophoresis/western blot analysis indicates that these Mabs are reactive with the HSC70 and HSP70 members of the rat HSP70 family. Immunohistological observations show that both Mabs are strongly reactive with a widely dispersed subpopulation of olfactory receptor neurons. Morphological, immunohistological, and autoradiographic birthdating analyses demonstrate that reactive cells are fully mature receptor neurons. Their reactivity, however, does not appear to be stress-related. More significantly, axons of reactive neurons show intense anti-2A4 reactivity. This has allowed us to trace these axons to their target glomeruli in the olfactory bulb, demonstrating that the reactive neurons project to just one to two glomeruli on either side of each bulb via consistent and predictable pathways. This is the first subpopulation of olfactory receptor neurons to be traced to such a small number of glomeruli. Given this extremely small number, it seems likely that the reactive receptor cell subpopulation serves some specific olfactory function. In addition, axonal 2A4 reactivity should also prove useful in defining the relative roles of receptor neurons and glomeruli in the establishment of epithelial-glomerular connections.

The dynamics of cell death in the olfactory epithelium.

Adult vertebrate olfactory epithelia are unique in their continual sensory neuron turnover and replacement. This paper describes studies of various aspects of the death of these receptor neurons in unoperated rats and at 12 days and 7 weeks after unilateral ablation of the olfactory bulb, the receptor neuron synaptic target. Particular attention has been focused on the lifespan of the dying cells using tritiated thymidine/autoradiography to examine their "birthdates." We show that in control epithelia 25-30% of the degenerating (pyknotic) cells were located in the basal quarter of the epithelium, the location of the least mature sensory neurons and of neuronal stem cell proliferation. Birthdate analysis shows that 2-5% of the degenerating cells were dying within a day or less of their "birth." Thus, a sizeable proportion of these cells were dying precociously, before achieving full neuronal maturation. A further 65% of the dying cells occurred in the middle half and 7% in the apical quarter of the epithelium. Following unilateral olfactory bulbectomy, a two- to threefold increase in the number of degenerating cells occurred in ipsilateral versus contralateral tissue. This was maintained through the 7-week experimental period. A shift of 10-15% of the total degenerating cell numbers from the basal to middle region of the epithelium also occurred. Despite the increased degenerative activity ipsilaterally, the proportion of dying cells labeled autoradiographically remained the same on both sides at most labeling periods. However, a striking wave of enhanced cell death of 6- to 7-day-old neurons over the contralateral levels occurred ipsilaterally in both the 12-day and 7-week postbulbectomy animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of ketamine on stress protein immunoreactivities in rat olfactory mucosa.

Administration of 9 mg ketamine per 100 g b.wt. to rats leads to transient enhancement of immunoreactivity to monoclonal antibodies against two stress proteins, ubiquitin and human 70 kDa heat shock protein (HSP70), in the supranuclear region of supporting cells of the olfactory epithelium and in the Bowman's gland acinar cells in the subepithelial lamina propria. In the supporting cells the enhanced immunoreactivities are not caused by other drugs used in our surgical anesthetic/antibiotic regimen (xylazine, buprenorphine, and gentamicin), but in Bowman's glands they are. Results are discussed in terms of possible ketamine binding to phencyclidine receptors (either NMDA-associated or not) and possible direct stress-inducing interactions of ketamine or ketamine breakdown products with the inhalant detoxification or secretory systems in the reactive cells.

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.

Rat olfactory neurons express a 200 kDa neurofilament.

Neurofilament expression in peripheral olfactory neurons of adult rats was investigated by immunoblotting and immunohistochemistry using monoclonal antibodies specific for each of the 3 neurofilament proteins. Immunoblotting analysis of olfactory epithelium extracts demonstrated the presence of only the 200 kDa (NFH) polypeptide; the 68 kDa (NFL) and 160 kDa (NFM) neurofilaments were not detected. Similarly, no immunoreactivity was observed in tissue sections using the NFL and NFM antibodies. In contrast, when sections were probed with the antibody to NFH, immunoreactivity was localized primarily in the dendritic knobs and near the cell bodies of the receptor cells.

Identification of a new non-neuronal cell type in rat olfactory epithelium.

We have examined adult and embryonic rat olfactory epithelia by immunohistochemical techniques using the monoclonal antibody 1A-6, which was raised against embryonic rat olfactory epithelia. A heretofore unidentified cell type, reactive with the monoclonal antibody 1A-6, was observed scattered within the epithelium. The 1A-6 reactivity of these cells is most intense on the microvilli projecting from the luminal cell surfaces. For several reasons, we believe these cells are not neurons but a distinct subpopulation of supporting cells or some other sort of non-neuronal cells. (1) They have no identifiable axonal process, are not reactive with an antibody against olfactory marker protein, and are not in juxtaposition with trigeminal axons. (2) They survive ablation of the olfactory bulb. (3) Their nuclei lie within the supporting cell layer, and they resemble supporting cells morphologically and in their [3H]thymidine birthdating and turnover characteristics. However, the 1A-6-positive cells fail to react with the general supporting cell-specific monoclonal antibody SUS-1 [see Hempstead J. L. and Morgan J. I. (1983) Brain Res. 188, 289-295] a finding which suggests that they are not typical supporting cells. Immunoreactivity to 1A-6 is developmentally regulated. Immunohistochemical preparations of almost all tissues we examined showed widespread reactivity in the embryo but a much more restricted pattern in the adult. In the olfactory epithelium of the fetus, the luminal surfaces of all cells, including supporting cells and olfactory receptor cells and cilia, are reactive, while in the adult only the non-neuronal cell subpopulation shows this reactivity. We also found that during the reconstitution of olfactory epithelium which occurs in response to olfactory bulbectomy-induced neuronal degeneration, fetal patterns of 1A-6 reactivity are not re-expressed, i.e. the only 1A-6-positive cells are the non-neuronal cells seen in unperturbed adult olfactory epithelium. Preliminary biochemical analyses of membrane fractions from E19 brain and from adult olfactory mucosa indicate that the 1A-6 reactivity is associated with two bands, having molecular weights of 42,000 and 46,000 on Western blots.

Developmental expression of reactivity to monoclonal antibodies generated against olfactory epithelia.

The developmental expression of immunocytochemical reactivity to 3 monoclonal antibodies (Mabs Neu 4, Neu 5, and Neu 9) that were generated against adult rat olfactory epithelium was examined in olfactory tissues of embryonic rats. Tissues examined included the nasal olfactory epithelium, nerve, and olfactory bulb, as well as vomeronasal epithelium and nerve. Reactivity patterns of these Mabs in adult rats have been described previously (Hempstead and Morgan, 1985a). All 3 Mabs show reactivity on the cell surfaces of neurons, axons, and dendrites of the olfactory epithelium proper. Neu 5 alone shows reactivity on the dendritic knobs, site of transduction of the olfactory stimuli. These reactivities appear early, suggesting developmentally significant roles for the antigens to these Mabs. For Neu 5 and Neu 9 initial reactivity occurs on outgrowing olfactory axons at E13. Dendritic and perikaryal reactivities begin appearing at E14. For Neu 4 initial reactivity occurs simultaneously on olfactory neuronal perikarya, axons, and dendrites at E14. Reactivity also occurs on cells that migrate from the olfactory epithelium and are associated with the olfactory nerves. Within the developing olfactory bulb, Neu 5 behaves as a general cell-surface marker. Neu 4 and Neu 9, however, show enhanced reactivity in the glomerular layer after the onset of synaptogenesis. Reactivity is also seen in the nasal respiratory epithelium and in the vomeronasal epithelia and nerve. Neu 5 and several antibodies to rat neural cell adhesion molecules (N-CAMs) show similar, although not identical, immunohistochemical staining patterns. They also react with the same bands in Western blots of brain membrane preparations. Western blots of Neu 5-reactive material also show developmental and spatial correlations of apparent molecular-weight distributions expected of N-CAM-like components as well.

Dorsal root ganglia development in chicks following partial ablation of the neural crest.

To assess the effects of reduced competition for peripheral targets on developing brachial dorsal root ganglia (DRG), chick embryos were subjected to partial ablations of the brachial neural crest at stages 13 or 14 (Hamburger, V., and H.L. Hamilton (1951) J. Morphol. 88: 49-92), using an ophthalmological cauterization unit. In the initial studies reported here, ganglia developing from the remaining crest material were examined for ganglionic volume and neuronal size, neuronal number, and degenerative activity at stage 35. Results showed that the lesion procedure resulted in the reduction or absence of one or two ganglia on each side at the level of DRG 15 to DRG 17. Hypertrophies occurred in other ganglia remaining at these and at more rostral levels and ranged up to 220%. These hypertrophies were most pronounced, however, not in the ganglia adjacent to those lesioned but rather in more remote ganglia, including those at cervical levels. Accompanying these ganglionic changes were significant alterations in all three neuronal parameters examined. The findings clearly demonstrate a responsiveness of chick brachial DRG to reduced competition resulting from neural crest ablations and that such responsiveness occurs along several axial segments.

Rapid appearance of labeled degenerating cells in the dorsal root ganglia after exposure of chick embryos to tritiated thymidine.

The interval between [3H]thymidine delivery and onset of cellular degeneration in 5.5 day embryonic chick brachial dorsal root ganglia was examined autoradiographically. Of the degenerating cells, 14% were labeled by 2 h after [3H]thymidine delivery. This percentage increased for 24 h. Wing bud amputation had no effect on this percentage through 9 h. Thus, some cause of cell death other than faulty peripheral connections may exist in at least some degenerating ganglionic cells.

Proliferative and degenerative events in the early development of chick dorsal root ganglia. I. Normal development.

Development of the chick dorsal root ganglia was examined in 4.5- to 9.5-day embryos. Tritiated thymidine (3H-TdR) and autoradiography was used to analyze proliferative activity and the Feulgen procedure to analyze degenerative activity in ganglia 12-17. Proliferative activity was found to be elevated through 4.5 days of incubation when as many as 14% of the ganglionic cells become labelled following a one-hour exposure to 3H-TdR. By 6.5 to 7.5 days proliferative activity decreases to 2-4% in the lateroventral (LV) regions and to approximately 1% in the mediodorsal (MD) regions of the ganglia. However, there appears to be increased proliferative activity by the end of the experimental period at 9.5 days. Birthdate studies demonstrate that large-scale neuronal production occurs between 4.5 and 6.5 days in the LV regions and between 4.5 and 7.5 days in the MD regions. After those times ganglionic proliferative activity must be largely nonneuronal in nature. This nonneuronal proliferation is greater in LV than in MD regions and in brachial than in nonbrachial ganglia. Degenerative activiy was found to be absent from the ganglia until after 4.5 days of incubation. It then increases rapidly, and by 5.5 days 5% of the LV cells in nonbrachial ganglia are degenerating. Degenerative activity then declines but is still present at 9.5 days. In contrast to results of an earlier study (Hamburger and Levi-Montalcini, '49), degenerative activity was also found in the LV region of brachial ganglia and the MD regions of brachial and nonbrachial ganglia. The pattern of LV degenerative activity in brachial ganglia is similar to that in nonbrachial ganglia, but the level of activity is lower. In the MD regions degenerative activity increases throughout the experimental period, and by 9.5 days as many as 4% of the MD cells are degenerating.

Proliferative and degenerative events in the early development of chick dorsal root ganglia. II. Responses to altered peripheral fields.

Responses of chick embryo dorsal root ganglia to early wing bud amputation were examined histologically using tritiated thymidine (3H-TdR) and autoradiography to analyze proliferation and the Feulgen procedure to visualize degenerating cells. Right wing buds were amputated at stage 15 or 16. At 4.5 to 9.5 days of incubation embryos were given a 1-hour exposure to 3H-TdR and fixed. Feulgen-stained autoradiographs were examined for percentage of cells labelled (labelling index) or degenerating (degeneration index) in lateroventral (LV) and mediodorsal (MD) regions of brachial (G14-16) and nonbrachial (G12, 13, 17) ganglia. The earliest response to amputation was a highly significant increase in degeneration indices of LV and MD regions of ipsilateral brachial ganglia at 5.5 days. Significant brachial LV responses were observed throughout the remainder of the experimental period. Two peaks occur in this response: at 5.5 days, corresponding to the peak seen in normal nonbrachial ganglia, and at 8.5 days, having no counterpart in normal development. In brachial MD regions significant degenerative responses occur at most times examined. Significant responses also occur at 7.5 and 8.5 days in MD regions of nonbrachial ganglia. The presence of MD responses in our material indicates that maturation of at least some MD neurons occurs earlier than previously thought. Significant labelling responses occur in brachial LV regions from 7.5 days on. Because other studies (Carr and Simpson, '78a) show that this time is after the end of large-scale neuronal production, this labelling response must be nonneuronal in nature. We conclude that this response is a secondary response to amputation, consequent to the greatly increased cellular degeneration. Results of experiments involving addition of limb buds at the brachial level are also presented.