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.

TGF-alpha and olfactory marker protein enhance mitosis in rat olfactory epithelium in vivo.

Previous studies demonstrated that both transforming growth factor-alpha (TGF-alpha) and olfactory marker protein (OMP) are potent enhancers of mitosis in fetal rat olfactory epithelium grown in organotypic culture. Here we show that when either of these two peptides is administered to adult rats they elicit a significant increase in uptake of tritiated thymidine ([3H]TdR) by olfactory epithelium. In addition OMP promotes an increase in uptake of [3H]TdR in liver, but TGF-alpha has no effect. The data argue that both peptides regulate the rate of cell division in rat olfactory epithelium in vivo and in vitro, and suggest there may be redundancy in the regulatory apparatus modulating cell division in this tissue.

Differential activation of ErbB receptors in the rat olfactory mucosa by transforming growth factor-alpha and epidermal growth factor in vivo.

Transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF) are members of the EGF family of growth factors. They have a common receptor, the EGF receptor. This belongs to the tyrosine kinase group of receptors called the ErbB receptor family. Other members are ErbB-2, ErbB-3, and ErbB-4. Binding of either ligand to the receptor elicits an increase in tyrosine kinase activity, resulting in the autophosphorylation of the receptor followed by a phosphorylation cascade of other tyrosine kinase substrates including mitogen-activated protein kinase (MAPK). TGF-alpha and EGF have been shown to stimulate cell division in the olfactory epithelium in vitro and may regulate cell division in vivo. To investigate whether exogenous TGF-alpha or EGF has a functional effect on the olfactory mucosa in vivo, 12.5-50 micrograms of each growth factor was administered to rats via the carotid artery. After 2 min, olfactory mucosa and liver samples were collected, homogenized, and immunoprecipitated with antibodies to the ErbB receptors. The immunoprecipitates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting. Using phosphotyrosine antibody, the receptors were probed for phosphorylation. Activation of MAPK was also investigated using MAPK antibody. Exogenous TGF-alpha activated EGFR, ErbB-2 and MAPK, whereas EGF activated only the EGFR. TGF-alpha was a more potent activator of EGFR than EGF. Neither ligand had an effect on ErbB-3 and ErbB-4 receptors. These effects were absent in the control animals which received the same solution without the growth factor. These results are consistent with the notion that binding of TGF-alpha to EGFR may play a role in olfactory cell division in vivo.

Inhibition and enhancement of odorant-induced cAMP accumulation in rat olfactory cilia by antibodies directed against G alpha S/olf- and G alpha i-protein subunits.

The odorant-induced accumulation of cAMP can be inhibited by antibodies directed against G alpha s/olf. In contrast, antibodies raised against G alpha i-subunits caused a strong enhancement of the odorant-induced cAMP accumulation. Western blotting and immunoelectron microscopy revealed the presence of both G alpha s/olf- and G alpha i-subunits in rat cilia preparations. The existence of both stimulatory and inhibitory odorant-induced regulation of adenylyl cyclase activity in olfactory cilia may indicate that an initial integration of different odorant stimuli begins at the level of primary reactions in the same effector enzyme.

Ultrastructural localization of amiloride-sensitive sodium channels and Na+,K(+)-ATPase in the rat's olfactory epithelial surface.

Several studies have indicated that olfactory responses are impeded by amiloride. Therefore, it was of interest to see whether, and if so which, olfactory epithelial cellular compartments have amiloride-sensitive structures. Using ultrastructural methods that involved rapid freezing, freeze-substitution and low temperature embedding of olfactory epithelia, this study shows that, in the rat, this tissue is immunoreactive to antibodies against amiloride sensitive Na(+)-channels. However, microvilli of olfactory supporting cells, as opposed to receptor cilia, contained most of the immunoreactive sites. Apices from which the microvilli sprout and receptor cell dendritic knobs had much less if any of the amiloride-antibody binding sites. Using a direct ligand-binding cytochemical method, this study also confirms earlier ones that showed that olfactory receptor cell cilia have Na+, K(+)-ATPase. It is proposed that supporting cell microvilli and the receptor cilia themselves have mechanisms, different but likely complementary, that participate in regulating the salt concentration around the receptor cell cilia. In this way, both structures help to provide the ambient mucous environment for receptor cells to function properly. This regulation of the salt concentration of an ambient fluid environment is a function that the olfactory epithelium shares with cells of transporting epithelia, such as those of kidney.

Regional distribution of rat electroolfactogram.

1. Electroolfactorgram (EOG) recordings were made from different regions of the rat olfactory epithelium to test for spatial distribution of odor responses. 2. The EOG recordings showed spatial distribution of the odor responses in the olfactory epithelium. While some odorants (amyl acetate, anisole, and ethyl butyrate) were more effective in evoking responses in the dorsal recess near the septum, other odorants (including limonene, cineole, cyclooctane, and hexane) were more effective in the lateral recesses among the turbinate bones. These differences were seen as statistically significant odorant-by-position interactions in analysis of variance. 3. Comparisons of recordings along the anteroposterior dimension of the epithelium produced smaller differences between the odor responses. These were not significant for 3-mm distances, but were statistically significant for 5- to 6-mm distances along the dorsomedial epithelium. 4. The latencies were significantly longer in the lateral recesses than in the medial region. This probably reflects a more tortuous air path along the turbinate bones to the lateral recesses. 5. The olfactory receptor cells were activated by antidromic stimulation via the nerve layer of the olfactory bulb. The population spikes evoked from the olfactory receptor cells could be suppressed by prior stimulation with odorants that evoked strong EOG responses. This collision of the antidromic action potentials with the odor-evoked action potentials indicates that the same population of receptor cells was activated in both cases. 6. The flow rate and duration of the artificial sniff were varied systematically in some experiments. The differential distribution of response sizes was present at all flow rates and sniff durations. Some odors (e.g., amyl acetate and anisole) produced increased responses in the epithelium of the lateral recesses when flow rates or sniff durations were high. We suggest that these changes may reflect the sorptive properties of the nasal membranes on these odors. The responses to other odors (e.g., hexane or limonene) were not greatly affected by flow rate or sniff duration. 7. Taken with existing anatomic data, the results indicate that the primary olfactory neurons that project axons to glomeruli in different parts of the olfactory bulb are responsive to different odors. The latency differences between responses at medial and lateral sites are large enough to be physiologically significant in the generation of the patterned responses of olfactory bulb neurons.

Organization of inhibition in the rat olfactory bulb external plexiform layer.

1. Intracellular recordings were made from the output neurons (mitral and tufted cells) of the rat olfactory bulb during electrical orthodromic stimulation of the olfactory nerve layer (ONL) and antidromic stimulation of the lateral olfactory tract and posterior piriform cortex (pPC) to test for physiological differences among the neuron types. Many of these neurons were identified by intracellular injections of biocytin, and others were identified by their pattern of antidromic activation. 2. Both marked and unmarked mitral cells showed large inhibitory postsynaptic potentials (IPSPs) in response to antidromic stimulation of the pPC, whereas tufted cells exhibited small IPSPs in response to pPC stimulation. Tufted cells, however, showed large IPSPs in response to ONL stimulation. In many cases, these tufted cell responses to ONL stimulation were larger than the mitral cell responses. The marked superficial tufted cells, those with basal dendrites in the superficial sublayer of the external plexiform layer (EPL), had the smallest IPSPs in response to pPC stimulation. These data support anatomic observations suggesting that the granule cell populations responsible for the IPSPs may be different for mitral and for superficial tufted cells. 3. The different types of output cells also showed differences in their responses to orthodromic stimulation. Type I mitral cells, which have basal dendrites confined to the deep sublayer of the EPL, were significantly less excitable by ONL stimulation than were the type II mitral cells, which have basal dendrites distributed within the intermediate sublayer of the EPL. Half of the type I mitral cells could not be excited at all by ONL stimulation. Superficial tufted cells showed even greater orthodromic excitability than type II mitral cells, usually responding to ONL stimulation with two or more spikes. 4. The ionic basis of the IPSPs in the superficial tufted cells appeared similar to those described for mitral cells. These IPSPs could be reversed by chloride injection and were associated with increased membrane conductance. 5. For both mitral and tufted cells, the number of ONL electrodes evoking IPSPs was greater than the number evoking spikes. These data suggest a kind of center-surround organization of inputs to these cells from the ONL, although this does not yet imply that the sensory receptive field of these output cells has a center-surround organization. 6. In conclusion, the properties of rat olfactory bulb output cells correlate with the sublayers of the EPL in which their basal dendrites lie.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of steroids on the olfactory function of the dog.

Twenty-four (24) mature, mixed breed, healthy dogs weighing from 14.6 kg to 27.6 kg were used to study the effects of various steroids on the olfactory function of the dog using olfactory detection threshold as an index. Two odorants were used, viz; benzaldehyde and eugenol. Of the various steroids used, only dexamethasone produced classical signs of Cushing's syndrome in the dogs. However, all dogs that received either dexamethasone alone or hydrocortisone plus DOCA exhibited a significant elevation in the olfactory detection threshold for both odorants without any observable structural alteration of the olfactory tissue using light microscopy. On the other hand, neither DOCA, hydrocortisone alone, nor any of the vehicles used in the study significantly altered the olfactory function of the dogs. The results show that Cushing's syndrome can be experimentally produced in dogs using exogenous steroids and that this condition diminishes the olfactory capability of the dog without producing classical signs of the disease.