Ultrastructural localization of olfactory transduction components: the G protein subunit Golfα and...

Neuron, Vol. 8, 441-453, March, 1992, Copyright 0 1992 by Cell Press

Ultrastructural localization of O lfactory Transduction Components: the G Protein Subunit Golfa and Type III Adenylyl Cyclase Bert P. M. Menco,* Richard C. Bruch,* Barbara Dau,* and Waleed Danho+ *Department of Neurobiology and Physiology Northwestern University Evanston, Illinois 60208-3520 +Peptide Research Department Hoffmann-La Roche, Inc. Nutley, New Jersey 07110

Summary

Electron microscopy and postembedding immunocytochemistry on rapidly frozen, freeze-substituted speci- mens of rat olfactory epithelia were used to study the subcellular localization of the transduction proteinsGolf, and type III adenylyl cyclase. Antibody binding sites for both of these proteins occur in the same receptor cell compartments, the distal segments of the olfactory cilia. These segments line the boundary between organism and external environment inside the olfactory part of the nasal cavity. Therefore, they are the receptor cell regions that most likely first encounter odorous com- pounds. The results presented here provide direct evidence to support the conclusion that the distal segments of the cilia contain the sites of the early events of olfactory transduction.

Introduction

It is generally assumed that olfactory cilia have membranes containing odorant receptors and the first parts of the apparatus needed to transduce odorous messages (Anholt, 1989,199l; Bakalyar and Reed, 1991; Bruch et al., 1988; Kinnamon and Getchell, 1991; Lan- cet, 1988; Lancet et al., 1989; Reed, 1990; Shirley and Persaud, 1990; Snyder et al., 1988). The transduction cascade begins when odorants interact with receptor molecules, probably members of the superfamily of G protein-linked receptors (Buck and Axel, 1991). The stimulus-receptor interaction leads to activation of a G protein, most likely the olfactory epithelium-specific G,lf,. This member of the family of guanine nucleotide-binding proteins shares extensive (88%) homology with G,, (Jones and Reed, 1989; Jones, 1990). It is hypothesized that Golfa in turn activates an adenylyl cyclase, again most likely a special type, called type III (Pfeuffer et al., 1989; Bakalyar and Reed, 1990). CAMP directly opens a cyclic nucleotide-gated ion channel (Dhallan et al., 1990; Ludwig et al., 1990) through which sodium can enter, thereby depolarizing the cell (Gold and Nakamura, 1987; Kaupp, 1991; Nakamura and Gold, 1987). Phosphodiesterase plays a role in the ter- mination of the signal by reducing free CAMP (Fire- stein et al., 1991). There are alternative olfactory signal transduction routes that work through cGMP instead of CAMP (in insects, see Ziegelberger et al., 1990) or

through G protein activation of phospholipase C and inositol trisphosphate-mediated calcium release (see also Anholt and Rivers, 1990; Bruch and Gold, 1990; Restrepo et al., 1990). Here we were concerned only with the CAMP-modulated cascade.

Recent biochemical and molecular cloning studies of Golfa and type III adenylyl cyclase have provided probes that allowed us to analyze the olfactory transduction apparatus with immunocytochemical methods at high resolution. Earlier immunocytochemical studies located G,, in the olfactory cilia; the common b subunits were found in cilia, but also in dendrites and dendritic endings (Mania-Farnell and Farbman, 1990). An ultrastructural enzyme histochemical study localized adenylyl cyclase to the olfactory cilia and dendritic endings (Asanuma and Nomura, 1991).

Light microscopy located Golfa (Jones and Reed, 1989) and type III adenylyl cyclase (Bakalyar and Reed, 1990) in the ciliated epithelial surface. Olfactory bul- bectomy abolished this labeling (Bakalyar and Reed, 1990; Jones and Reed, 1989), suggesting that receptor cell structures, most likely cilia, were labeled under normal conditions. However, fine-structural localization was needed to provide direct evidence as to whether Golfa and type III adenylyl cyclase occur in the same cellular compartments; whether these compartments are cilia, dendritic endings, or both; and whether these proteins were uniformly or regionally distributed along the cilia. To these ends, we used ultrastructural cytochemical methods under conditions of optimal preservation-rapid freezing, freeze- substitution, and low temperature embedding (Kel- lenberger, 1991; Menco, 1989a; Phillips and Bridgman, 1991)-to study the ultrastructural localization of transduction proteins in the olfactory receptor cells.

Results

lmmunoblots Antibody specificity was evaluated in preparations of rat olfactory cilia by immunoblotting. Antibodies to Golfa (Jones and Reed, 1989) and type III adenylyl cyclase (Bakalyar and Reed, 1990) labeled single bands of appropriate molecular weight on Western blots, 45,000 for Golfa and about 200,000 for type I I I adenylyl cyclase. Golfa immunoreactivity was completely abolished by prior incubation of the antibody with its anti- genie peptide.

Light Microscopic lmmunocytochemistry Using light microscopy, antibodies to C&, (see also Jones and Reed, 1989) and type III adenylyl cyclase (Bakalyar and Reed, 1990) clearly labeled the olfactory epithelial surface, whereas the respiratory epithelial surface was not labeled. The contrast is especially clear in the transition zone between the two epithelia (Figures laand lb). However, atthis level of magnifica-

NelJKX 442

: “$

. : . : ,

0 lb

Figure 1. Labeling of Rat Nasal Epithelia with Olfactory Transduction Antibodies: Light Microscopy

Labeling with antibodies for G,,,(a) and for type III adenylyl cyclase (b) was continuous over the olfactory epithelial surface (to the right of the arrowheads) and absent over the respiratory epithelial surface (to the left of the arrow). Bar, 20 wm.

tion, it was difficult to decide which cell surface compartment(s) was labeled. Electron microscopy was therefore performed to identify the cellular sites of antibody binding at high resolution.

Electron Microscopic lmmunocytochemistry The long and thin distal parts of the rat’s olfactory cilia bound more of both primary antibodies-those to Gorra and those to type III adenylyl cyclase-than the proximal parts of the cilia, the dendritic endings where the cilia originate, and the microvilli of neighboring support cells. This was the case irrespective of whether protein C (Figures 2aand 2b; Figure 3; Figure 4) or goat anti-rabbit antibodies (data not shown) conjugated to colloidal gold were used as secondary probe. Otherwise, neither the dendrites themselves, nor any other structure labeled within regions of good tissue preservation. Other structures included apical regions of infrequent microvillous cells (Carr et al., 1991; Menco, 1992b). Thedistribution of the gold particles was not homogeneous; some distal parts of the olfactory cilia contained more labeling than others; this was true for antibodies to both transduction proteins (see Figures 2a and 2b). Also, the intensity of labeling with undiluted antibodies and antibodies diluted I:4 was similar. The latter dilution was used as comparison for immunoadsorption controls (see Controls).

We made semiquantitative evaluations of the results in cilia, dendritic knobs, and supporting cell microvilli. Antibodies to G,I,~ and those to type ill adenylyl cyclase displayed the same relative pattern of labeling in all cellular compartments considered. Den- dritic endings and supporting cell microvilli had 20%- 40% of the labeling of the distal parts of the cilia, whereas the proximal parts of the cilia labeled somewhat more intensely, 30%-50% relative to the distal parts of the cilia. Thus, there may have been a slight gradient from receptor cell dendritic endings to cilia, with the proximal partsof the ciliaexpressing interme- diate levels of antigen. However, statistically, only the differences between thedistal parts and all othercom- partments considered-the proximal parts of cilia, receptor cell dendritic endings, and supporting cell microvilli-were significant (p < 0.01 according to Fisher’s least significant difference test).

Absolute values of densities of labeled gold particles were, unlike relative ones, not meaningful. Val- ues of 15-25 gold-labeled particles per urn2 in the distal parts of the olfactory cilia were probably under- estimates for the numbers of antigen-containing moi- ecules. (Quantification was done only for sections labeled with protein G as secondary probe. Goat anti- rabbit antibodies conjugated to gold served merely as a control to determine whether the pattern of labeling was the same with a different secondary probe.) Phil-

Localization of Olfactory Transduction Components 443

Figure 2. Labeling of Olfactory Receptor Cell Structures with Olfactory Transduction Antibodies: Electron Microscopy

Dendritic ending structures (E) of a rat olfactory receptor cell labeled with polyclonal antibodies to a subunits of C,I~. (a) and type III adenylyl cyclase (b) visualized with protein C conjugated to 10 nm gold particles, as secondary probe. The thin distal parts of the cilia (D) labeled more intensely than the proximal parts (P) and the dendritic endings. Supporting cell microvilli (S) show some scattered labeling. Bar, 0.5 urn.

Figure 3. Olfactory Cilia: Distal Parts

Higher magnification of the distal parts (D) of the olfactory cilia that labeled with affinity/-purified antibodies to r&,. Supporting cell microvilli (S), usually oriented perpendicular to the cilia, did not exhibit labeling. At this magnification, antibodies to type ill adenylyl cyclase displayed a similar pattern of labeling. Bar, 0.5 Km.

Figure 4. Schematic Representation of the Major Components of the Mammalian Ol- factory Epithelial Surface

Receptor cell dendrites (DE) terminate in dendritic endings (E), from which IO-20 olfactory cilia sprout. These cilia consist of thick proximal parts (P), internally having complete axonemes, and of much longer (~50 flm) distal parts (D), with reduced in- ternal axonemal configurations. The distal cilium segments near the nasal lumen (NL) are from neighboring receptor cells. The receptor cells are surrounded by supporting ceils (S), which have many microvilli (MV) in their surfaces. The course of most of these microvilli is perpendicular to that of most of the distal parts of the cilia. Den- dritic endings, cilia, and microvilli are embedded in a mucus layer (MU), which bor- ders the nasal lumen (see, e.g., Menco, 1980a, for more details). The distal parts, hatched here, of the olfactory cilia display most of the antigenicity toward antibodies against the transduction proteins.


Figure 5. Respiratory Controls

Rat nasal respiratory ciliated surface labeled with polyclonal antibodies to u subunits of the olfactory C protein, CO,,. (a) and type III adenylyl cyclase (b) visualized with protein C, conjugated to 10 nm gold particles, as secondary probe. Unlike olfactory cilia, respiratory cilia did not display specific antibody binding, but only some scattered labeling (compare with Figures 2 and 3). Bar, 0.5 urn.

lips and Bridgman (1991) calculated a labeling efficacy of 3%-13% for seven different monoclonal antibodies to the same antigen, the nicotinic acetylcholine receptor. They compared their electron microscopic cytochemical resultswith those from biochemical binding studies. The polyclonal antibodies used here were made against small peptide regions, resembling in that respect monoclonal antibodies, which also tend to bind to a restricted region of larger molecules. Oth- erwise, the ultrastructural experimental conditions used here were essentially the same as the ones used by Phillips and Bridgman (1991). Also, the stoichiometry of G proteins and adenylyl cyclase is probably not l:l, as the absolute values would suggest, but could even be around 3O:l (Alousi et al., 1991). Thus, unless one knows more characteristics of the antibodies and antigens, relative and proportional values rather than absolute counts of numbers of labeled particles are more meaningful.

Controls We used several controls. Antibodies to neither Golfa nor type III adenylyl cyclase bound markedly to respiratory cilia (Figures la and lb, light microscopy; Fig-

ures 5a and 5b, electron microscopy). Immunoadsorp- tion of Golfa and type III adenylyl cyclase antibodies with an excess of their respective peptides before incubation of sections completely abolished labeling at both light (data not shown) and electron (Figures 6a and 6b) microscopic levels in either case. Incubation with normal rabbit serum instead of primary antibody solutions gave no specific labeling. Also, the Gorr,, peptide did not abolish labeling of antibodies to the C-terminal peptide of G,, (Simonds et al., 1989), which shares most of its amino acid sequencewith Golfa (data not shown).

Antibodies to two other receptor cell antigens gave a pattern of labeling which differed from that of the transduction proteins. The antigens were olfactory marker protein (OMP; Margolis, 1972, 1982) and 8-tubulin (Burton, 1992). Within regions of adequate to good freeze-fixed tissue preservation, the polyclonal anti-OMP antibodies labeled dendrites and dendritic endings throughout and, more sparsely, olfactory cilia (Figure 7a; see also Menco, 1989b). A monoclonal antibody against fi-tubulin outlined microtubules in all cellular compartments, dendrites (data not shown), dendritic endings, including their basal bodies, and

Figure 6. lmmunoadsorption Controls

Dendritic ending structures (E) of a rat olfactory receptor cell labeled with antibodies to G,ll, (a) and type ii/ adenylyl cyclase (b) immunoadsorbed totheir peptideantigens. Protein G, conjugated to 10 nm gold particles, was used as secondary probe for visualization. D, distal parts of the olfactory cilia; P, proximal parts of the olfactory cilia; S, supporting cell microviili. Bar, 0.5 pm.

olfactory cilia (Figure 7b), as well as respiratory cilia and their basal bodies (data not shown). Although anti-OMP and anti-fi-tubulin antibodies labeled dendritic endings and the proximal parts of the olfactory cilia much more strongly than either antibody to the transduction proteins, labeling of the distal parts of the cilia was less intense and rather diffuse with both antibodies. In these distal parts, the difference in labeling of control (OMP and fi-tubulin) and experimental (Golfu, type III adenylyl cyclase) antibodies was not very large. The low intensity of labeling in the distal parts of the cilia may be caused by the fact that these were often narrower than the tissue sections and, therefore, not completely accessible to the antibody solutions.

However, the major conclusion to be drawn from the above is that the distal parts of the olfactory cilia were best labeled with antibodies to the transduction proteins and that antibodies to other, unrelated proteins labeled other cellular compartments equally well, if not better, than the cilia (compare Figures 2a and 2b with Figures 7a and 7b).

Discussion

Olfactory Signal Transduction and the Morphology of Olfactory Cilia A major advantage of the ultrastructural techniques used in this study was that the olfactory epithelial surface, including the delicate, thin distal parts of the olfactory cilia, waswell preserved (Menco, 1989a). This allowedexposureofthesecellularcomponentsto(immuno)cytochemical treatments.

It is generally assumed that odorous messages are transduced, coded, and transported to the brain, where they are decoded, to initiate the organism’s appropriate response. Here we localized the cellular sites of initial signal transduction and showed that compartmentalization of the transduction apparatus parallels the morphological division in the thick proximal and the thin distal parts of mammalian olfactory cilia (Figure 4).

Proximal parts of mammalian olfactory cilia are 2-3 pm long. Like other cilia, including motile ones equippedwithacomplete9(2)+2setof microtubules,


Figure 7. Positive Controls Rat olfactory receptor cell dendritic endings labeled with polyclonal (rabbit) anti-OMP antibodies (a) and monoclonal anti-g-tubulin antibodies (b). Anti-OMP antibodies labeled dendrites (not seen here), their endings, and somewhat less prominently, their cilia. Anti-g-tubulin outlined microtubular structures of the dendritic endings and cilia, including basal bodies (b). For both antibodies, labeling of the distal parts of the cilia (D) was diffuse compared with that of the dendritic endings and proximal parts of the cilia (P). Binding was visualized with protein G-conjugated gold in (a); for the anti-g-tubulin monoclonal antibodies, goat anti-mouse IgGllgM conjugated to gold was used as secondary probe. The gold particles had a diameter of IO nm in either case. S, supporting cell microvilli. Bar, 0.5 urn.

they are about 0.28 pm across. On the other hand, the toreceptor cell outer segments, rods and cones, in morphology of the distal parts of the olfactory cilia that they are specially modified regions of cilia that differs drastically from that of motile cilia. They are serve a sensory function (Besharse and Horst, 1990; long, 50-60 pm, and about 0.11 pm across and usually Duncan, 1977; Vinnikov, 1982). have only two, sometimes one, microtubules (Lidow This study provides direct evidence that the mor- and Menco, 1984; Menco, 1984, 1992a; Seifert, 1970). phological specialization of the olfactory cilia is ac- The modified partsof theolfactorycilia resemble pho- companied by a molecular, and therefore most likely

also functional, compartmentalization. We showed that the distal parts of the olfactory cilia are the regions containing most of the sites of initial olfactory transduction. This is in line with the notion that the distal parts are likely to meet and interact with odor- ouscompoundsfirst. In an earlier, physiological study it was inferred that transduction occurs mainly in or near the receptor cell dendritic endings and proximal parts of the cilia (Getchell et al., 1980). In a recently published study, Lowe and Gold (1991) gave a possible explanation forthediscrepancythattheyattributed to an intrinsic latency of olfactorytransduction of several hundred milliseconds.

The rather irregular distribution of gold particles (Figure 2) along the distal parts of the olfactory cilia paralleled freeze-etch observations; portions of cilia in the same replica, next to each other, can have dif- fering amounts of intramembrane particles (see, e. g., Figure 13 in Menco, 1984). This heterogeneity may not be noticeable electrophysiologically since, at least in the salamander, the electrophysiological respon- siveness toward odorants was on average the same throughout thecilia(Loweand Gold, 1991). The results of Lowe and Gold also seem to be inconsistent with the difference between the proximal and distal parts of the olfactory cilia reported here. This discrepancy may reflect the different species used, but it might also be difficult to distinguish the shorter proximal parts of the cilia from the much longer distal parts with electrophysiology. Finally, unlike C& and type III adenylyl cyclase, the channels, eventually responsi- ble for the electrophysiological response may be ho- mogeneously distributed over the cilia.

c olfa

CTP-binding proteins couple the olfactory receptors to the rest of the transduction cascade, in particular adenylyl cyclase (Anholt et al., 1987; Bruch and Kalinoski, 1987; Novoselov et al., 1988; Pace and Lancet, 1986; Parfenova and Etingof, 1988). The olfactory epithelium has a unique stimulatory G protein subunit, Golfa, which has been cloned (Jones and Reed, 1989). The fact that Golfo and G,, subunits share 88% sequence homology (Jones and Reed, 1989) may underlie the observation that antibodies to several G,, peptides cross-reacted with Golfa (Jones, 1990) and displayed cytochemical labeling patterns resembling those of Golfa, with both light and electron microscopy. In contrast, the antibodies to Golro used in this study did not cross-react with G,,. We have evidence suggesting that antibodies to Golf0 and G,, label differently indeed; antibodies to G,, peptides labeled olfactory epithelial surface structures at least 2 days before Golfo antibodies during embryonic development (Mania-Farnell and Farbman, 1990; B. Dau, B. P. M. Menco, R. C. Bruch, W. Danho, and A. Farbman, 1991, Chem. Senses, abstract; unpublished data).

In earlier freeze-substitution studies we showed that the narrow cytoplasmic matrix surrounding the microtubules of distal parts of the olfactory cilia is

electron-opaque, especially after osmium fixation (Menco, 1984, 1986). This suggests that these parts of the cilia contain significant quantities of protein. Except for (j-tubulin (Figure 7b), OMP was the only protein known to be present inside the cilia (Figure 7a). With antisera to OMP generated in both goat (Menco, 198913) and rabbit (Figure 7a), we showed that the cilia were not as heavily labeled as the dendritic endings and dendrites. The reverse is true for G,,,,, suggesting that a considerable part of the ciliary matrix proteins might correspond to G proteins, which are partly immersed in the inner membrane leaflet (Gilman, 1987; Roof and Heuser, 1982; Spiegel et al., 1991).

Type III Adenylyl Cyclase An important role for CAMP in olfaction was implied by the electro-olfactogram (EOG) recordings of Minor and Sakina (1973) and Menevse et ai. (1977). CAMP binds to a novel, cyclic nucleotide-gated ion channel, which subsequently opens to increase the sodium conductance. This eventually leads to depolarization of the receptor cell, as shown in many recent studies (Bruch and Teeter, 1990; Firestein et al., 1991; Frings and Lindemann, 1991; Gold and Nakamura, 1987; Kolesnikovet al., 1990; Kurahashi, 1990; Kurahashi and Kaneko, 1991; Kleene and Gesteland, 1991; Labarca and Bacigalupo,l988; Loweand Gold,1991; Nakamura and Gold, 1987; Suzuki, 1989; Trotier et al., 1989; Vody- anoy, 1991). The presumptive cyclic nucleotide-gated channels have been cloned (Dhallan et al., 1990; Lud- wig et al., 1990).

The cyclic nucleotide activating these channels is generated by adenylyl cyclase from ATP (Pfeuffer et al., 1989), and the olfactory epithelium has a high adenylyl cyclase activity (Kurihara and Koyama, 1972). Odorants stimulate this activity, especially in the presence of GTP, not only in membrane preparations of olfactory epithelia (Shirley et al., 1986), but also in cell cultures (Ronnett et al., 1991). The activity was restricted mainly to preparations of isolated cilia (O’Connell et al., 1990; Pace et al., 1985; Sklar et al., 1986) and was absent in membrane preparations of deciliated epithelia (Chen et al., 1986). Nonhydrolyz- able GTP analogs, such as GTPyS, uncouple the odorant activity (for review see Lancet et al., 1989). There is a strong correlation between adenylyl cyclase activity and electrophysiological activity, as indicated by both EOGs (Lowe et al., 1989) and single unit recordings (O’Connell et al., 1990), and between adenylyl cyclase activity and human psychophysical responses (Doty et al., 1990). A novel type Ill adenylyl cyclase is present in olfactory neurons and has been cloned (Bakalyar and Reed, 1990; these authors used the label “III” to distinguish this adenylyl cyclase from two other types of this enzyme that occur especially in the brain). This form of the enzyme is likely to be ihe one stimulated byodorants (Lazard et al., 1989). Antibodies generated against a peptide of rat type III adenylyl cyclase were used in this study to localize the enzyme to a specific


fine structure (Bakalyar and Reed, 1990); antibody binding sites for the enzyme occurred in the same cellular compartment as those for G,lt,.

An earlier ultrastructural study, which made use of enzymatic cytochemical methods, also suggested that adenylyl cyclase was located primarily in the distal parts of rat olfactory cilia (Asanuma and Nomura, 1991). The present study, based on immunocytochemistry, suggests that it was type III adenylyl cyclase that was in part detected in that study. With enzyme cytochemistry there was also a microtubule-associated precipitate, which was absent in our analysis. In a fish, the Baikalgrayling,theenzyme histochemical method localized the enzyme to apical regions of both ciliated and microvillous olfactory receptor cells, including sensory cilia and microvilli. Finer distinctions were not determined (Pyatkina and Dmitrieva, 1990).

Although distal parts of the olfactory cilia were prominently labeled with antibodies to type III adenylyl cyclase, the results do not permit a clear verdict on whether the labeled structures were membranes or cytoplasmic compartments. Section thickness was usually greater than the diameters of the distal parts of the cilia, about 0.2 pm and 0.1 pm, respectively (Menco, 1989b); large portions of distal parts were often completely contained within the sections and were inaccessible to postembedding cytochemistry. Even labeling of cytoplasmic proteins, OMP (Figure 7a) and tubulin (Figure 7b), was more diffuse in the distal parts of the cilia than in their proximal parts and the dendritic endings. The last two structures traverse the thickness of the section thickness. Also, when membranes grazed the section surface, cytoplasmic components of the cilia may appear to be labeled rather than their membranes. But, from biochemical studies we know that adenylyl cyclase is a transmembrane protein (Krupinski et al., 1989).

Freeze-fracture particles of the olfactory cilia (Menco, 1980b, 1984) could, at least in part, represent the transmembranous type I II adenylyl cyclase. Many of the proteins reflected as particles bound the lectin wheat germ agglutinin (WGA; Menco, 1992b), which also inhibited both the electrophysiological response to odorants and the adenylyl cyclase activity of olfactory epithelia (Dr. E. H. Polak, personal communica- tion). Furthermore, adenylyl cyclase bound WGA (Krupinski et al., 1989). However, there were many more WGA-binding sites (Menco, 199213) than sites labeled with antibodies to type III adenylyl cyclase (Figure 2b). With protein G as a secondary probe, we found only about 25 gold-labeled particles per pm2 in the distal parts of the olfactory cilia in thin sections; with goat anti-rabbit antibodies conjugated to gold, this density was somewhat higher. In contrast, these plus dendritic endings and proximal parts of the cilia had about 600-700 WGA-labeled particles per pm2 in deep-etched, freeze-fractured replicas (Menco, 1992b). If our labeling efficacy was in the same range as that reported by Phillips and Bridgman (1991) (3%- 13%) for acetylcholine receptors, around 75-325 of

the transmembrane proteins could have represented type III adenylyl cyclase. Lancet and colleagues calculated that about 0.1% of the total ciliary protein, or 1% of their membrane proteins, could beadenylyl cyclase (Lancet et al., 1989; Pfeuffer et al., 1989). In earlier studies we calculated that there are about 1000-2500 intramembrane particles per pm2 (summarized in Menco, 1992a), which would give a smallest value of about IO-25 type III adenylyl cyclase molecules per pm2. Either calculation suggests that the total number of transmembrane proteins reflected as freeze-fracture particles is larger than the total number of adenylyl cyclase molecules, and many particles probably represented other transmembrane proteins, e.g., pu- tative receptors (Buck and Axel, 1991) and ion channels (Gold and Nakamura, 1987; Kurahashi and Ka- neko, 1991; Labarca and Bacigalupo, 1988; Nakamura and Gold, 1987).

Conclusion Two transduction steps occur after odor molecules interact with their receptors: the activation of a G protein and the subsequent activation of an adenylyl cyclase. Calfa and adenylyl cyclase were specifically present in apical regions of olfactory epithelia (Bak- alyar and Reed, 1990; Jones and Reed, 1989). Although physiological evidence for the specific involvement in olfactory transduction of these two proteins is still lacking, this study supports the notion that such involvement is rather likely. We showed that Golfa and type III adenylyl cyclase were localized mainly in the distal parts of the olfactory cilia. In the visual system, the equivalent G protein subunit, a-transducin, occurs in the cellular compartments where the interaction of light with the photoreceptor molecule, rho- dopsin, takes place, i.e., the outer segment discs (Roof and Heth, 1988; Roof and Heuser, 1982; van Veen et al., 1986). Like photoreceptor cell outer segments, olfactory receptor cell cilia are a modified type of cilium (see Figure 4), assuming a shape distinctive to their particular sensory function (Vinnikov, 1982). Based on the above analogies, it is reasonable to assume that the first proteins of the olfactory signal transduction cascade, the receptors (Buck and Axel, 1991; Lowe and Gold, 1991), are also located mainly in the distal parts of the olfactory cilia. These distal parts of the cilia increase the membranous receptor apparatus-containing surface of the epithelium about 40-fold (Menco, 1980a). This surface is distributed over many receptor cells, enabling the system to expose many receptors to the external odorous environment and thus vastly increasing the possibility of odor discrimination. The transduction proteins localized in this study are important in the subsequent signal processing.

Experimental Procedures

Animals, Fixation, and Freezing Procedures Olfactory and respiratory epithelia of nasal septa were obtained from young Sprague-Dawley rats (Harlan, Inc., Indianapolis, IN).

For light microscopy, animals were deeply anesthetized with 0.85 mglkg sodium pentobarbital, injected intraperitoneally, and fixed by transcardial perfusion with 4% paraformaldehyde. The tissues were removed and further fixed in the same fixative for several hours or overnight, followed by decalcification in RDO (Apex Engineering Corp., Plainfield, IL) for 4-5 hr. Embedding was carried out in Tissue-Tek OCT cryo-embedding compound (Miles, Elkart, IN). Cryostat sections, 10 pm thick, were cut with a Leica-Reichert Frigocut28OON Cryostat (Heidelberg, Germany) and were mounted on gelatin-and alum-subbed slides.

For electron microscopy, the rats were asphyxiated with CO,. The nasal septa were removed, mounted on aluminum disks, frozen on a liquid nitrogen-cooled copper block with the bounce-free Gentleman Jim quick-freeze system (Pelco, Inc., Tustin, CA; Heuser et al., 1979; Phillips and Boyne, 1984), and stored in liquid nitrogen until further use.

Antibodies and Antigens A monoclonal antibody to 8-tubulin (sea urchin), used as a positive control, was obtained from East-Acres (Southbridge, MA). Anti-rabbit OMP was a gift from Dr. F. L. Margolis (Roche Insti- tute of Molecular Biology, Nutley, NJ). Affinity-purified polyclonal antibodies raised in rabbits to peptide fragments RR3 (amino acids 8-22: (CY)KTAEDQGVDEKERREA) of C,,rf, (DJ6.3AP) and type III adenylyl cyclase (anti-HAB-I against amino acids 1106-1121: (H,N-Y)PNGSSVTLPHQVVDNP-COOH) and the peptide fragments themselves were kindly supplied by Drs. R. R. Reed and H. A. Bakalyar (Howard Hughes Foundation, Johns Hopkins University, Baltimore, MD; Bakalyar and Reed, 1990; Jones and Reed, 1989). In addition, polyclonal antibodies to &I,,, also raised in rabbits,were independently obtained byconjugat- ing the G,,r, peptide sequence RR3 without the CY dipeptide to bovine thyroglobulin with glutaraldehyde. Affinity purification of the antibodies was performed on a peptide column as pre- viously described (Jones and Reed, 1989). Antibodies to a C,, peptide (C-terminal decapeptide: RMHLRQYELL; Simonds et al., 1989) were supplied by New England Nuclear (RMII; catalog number NEI-805, lot number PAOII; New England Nuclear Re- search Products, DuPont, Boston, MA).

lmmunoblots Ciliary preparations of olfactory epithelia from adult rats were obtained as described earlier (Boyle et al., 1987). The deciliation medium contained IO mM Car&, 10 pgiml leupeptin, and 0.3 mM sucrose in 50 mM ACES (N-(2.acetamido))2-aminoethanesulfonic acid) in NaOH. Isolated cilia were resuspended to a protein con- centration of about 0.2 mg/ml. Protein content was determined with the Bradford (1976) method, with bovine serum albumin (BSA) as a standard. Cilium fractions were resolved in 8% or 10% SDS-polyacrylamide gels and transferred to Westran membranes (Schleicher and Schuell, Keene, NH) as described earlier (Bruch and Kalinoski, 1987). Immunoblots, probed with each antibody, were processed as described by Bruch and Carr (1991).

Freeze-Substitution and Lowicryl KllM Embedding Freeze-substitution and Lowicryl KIIM embedding werecarried out as described elsewhere (Edelmann, 1988; Menco, 1989a, 199213; Phillips and Bridgman, 1991). Briefly, infiltration and low temperature embedding were done in a CS Auto cryo-substitution apparatus (Leica-Reichert Instruments, Vienna, Austria). Neither aldehyde nor OsOl fixation was used. For embedding, we used Lowicryl KIIM (Chemische Werke Lowi GmbH, Wald- kraiburg, Germany). This hydrophilic resin allows infiltration and embedding at low temperatures. Hardening of the resin was done by exposure to ultraviolet light (Carlemalm and Villiger, 1989). The total time used for substitution at -80°C, infiltration at -80°C to -60°C, and embedding at -6OV to +25OC was about 1 month.

Light Microscopy: Postembedding lmmunocytochemistry The sections were preincubated in 0.1% SDS for 20 min at room temperature and then in normal goat serum from thevectastain ABC (see below) kit for 30 min. Next, they were reacted with

primary antibody in phosphate-buffered saline, supplemented with 0.1% BSA, for 1 hr at 37OC at proper dilutions, 1:60 for C,,,, and I:100 for type I!I adenylyl cyclase. Antigen reactivity was visualized with secondary antibodies conjugated to biotin followed by an avidin-biotin-peroxidase reaction using rabbit ABC kits (Vector Labs., Burlingame, CA). Diaminobenzidine served as the chromophore (see, e.g., Carr et al., 1989).

Electron Microscopy: Postembedding lmmunocytochemistry Sections of olfactory and nasal respiratory epithelia, gold to pur- ple in color, were collected on the rough side of uncoated nickel grids (200 or 300 mesh, thin bar hexagonal). Most of the postembedding procedures (preincubation, gold conjugate labeling, and contrast staining) were carried out at room temperature in moistchambersconsistingofTeflondisheswith60circularnum- bered welis, each about 0.5 cm in diameter and 0.25 cm deep. The dishes were covered with glass petri dish tops and wrapped in Parafilm lo prevent evaporation.

All procedures related to antibody binding were carried out on both sides of the grids. \Ne used various procedures for blocking and various dilution of antibodies. All of these gave similar results. Most commonly, we used TBST (0.01 M Tris, 500 mM NaCI, 0.1% Tween 20 [pH 8.01) supplemented with 3.6% BSA, 0.4% gelatin, and 10% normal horse serum for blocking and TBST supplemented with 0.1% gelatin, 0.9% BSA, and 0.1% Tween 20 for antibody dilution. More recently, we used the same buffer, supplemented with 0.1% acetylated BSA (Aurion, Wageningen, The Netherlands), but without Tween 20, for both blocking and antibody dilution (Leunissen, 1990). Blocking lasted 2-3 hr. Next, the sections were incubated overnight at 4OC in undiluted primary antibody solutions or in dilutions up to 1:4, without inter- mediary wash. This was done in capped 200 PI microcentrifuge tubes (Robinson Scientific, Sunnyvale, CA) tr immed to about 100 ~1. In this way, evaporation did not occur, even when the grids were immersed in volumes of antibody solution as small as 20 ~1 over times as long as 72 hr.

Binding ofthe primary polyclonal antibodies was determined with protein G (Biocell, Cardiff, UK,orAmersham, Inc.,Arlington Heights, IL; Bendayan and Garzon, 1988) or goat anti-rabbit secondary antibodies (Aurion) conjugated to 10 nm colloidal gold; gold-conjugated (IO nm)goatanti-mouse IgCilgM (Biocell or Am- ersham) was used for the detection of binding of the anti-p- tubulin monoclonal antibody. Gold soiswerediluted in thesame buffer used by the manufacturer (0.02 M Tris, 150 mM NaCi [pH 8.2]), supplemented with 1% BSA or 0.1% acetylated BSA and 0.1% Tween 20. We used optical densities of 0.20 at wavelengths of 520 nm for all gold probes.

Controls The following controls were used: -To test antibody specificity for G,,,, and type III adenylyl cyclase, the corresponding peptide antigens were used at respective concentrations of 1 mg/ml and IO mgiml with antibody concentrations of 0.05 mgiml (C&I,,) and 0.4 mgiml (type Ill adenylyl cyclase) in immunoadsorption experiments. This meant that the antibody solutions were diluted 4 times, compared with the stocks. The G,, antibody, RMII, was also incubated with an excess of C,rc, peptide. Peptide-antibody mixtures were left to stand for 24 hr at 4°C. After preincubation with blocking reagents, sections were incubated in the immunoadsorbed antibody solutions, antibodies diluted in the same buffer but without peptide (experimentals), and peptides without the antibodies. For light microscopy, the above mixtures were further diluted with phosphate-buffered saline so that the same final antibody concentrations as those listed under Light Microscopy: Postembedding lmmunocytochemistry were obtained. -Normal rabbit sera served as negative controls for immunoreactivity. - Nonsensoryciiia of the nasai respiratoryepithelium were used for comparison. -Antibodies to antigens present in the olfactory cilia, OMP (Farbman and Margolis, 1980; Menco, 1989b) and B-tubulin (Bur- ton, 1992), were used as controls to compare their patterns of


labeling with those of antibodies to the transduction proteins. The control antibodies were used at dilutions of I:6 (anti-p- tubulin) and I:5 (anti-OMP).

Electron Microscopy After jet washing in the proper buffer and distilled water, each for about 1 min, the sections were dried in a dessicator, stained with filtered 0.5% uranyl acetate in 50% methanol, carbon coated as described (Menco, 1992b), and examined at 120 kV in a JEOL 100 CX Temscan electron microscope. Both staining and carbon coating were done on the section-containing sides only.

Quantification of Results Semiquantitative evaluations were performed. These were de- rived from 12-56 observations of micrographs in which a particular structure was present. Counts of numbers of gold-labeled particles were carried out at final magnifications of 14,000x- 48,000x, but usually at 19,000x with a grid ruled in 1 cm> divi- sions. Particles over ciliary and microvillar compartments were counted, includingthosethatwere0.1 vmawayfromthesecom- partments and from membranes of dendritic endings. A one-way analysis of variance (ANOVA) and individual comparisons with Fisher’s least significant difference test were carried out with a Macintosh SE30 personal computer and the Statview II (Abacus Concepts, Berkeley, CA) statistical package.

Acknowledgments

Drs. Heather A. Bakalyar, Randall R. Reed (Department of Molec- ular Biology and Genetics, The Howard Hughes Medical Insti- tute, Johns Hopkins University School of Medicine, Baltimore, MD), and Frank L. Margolis (Roche Institute of Molecular Biol- ogy, Nutley, NJ) are thanked for sharing their antibodies. Dr. Ernest H. Polak (Laboratoire de Neurophysiologie, Universite Pierre et Marie Curie, Paris, France) is thanked for letting us use some of his unpublished data. Mr. Eugene W. Minner’s helpwith photography was greatly appreciated. This work was supported by grants from the NSF to B. P. M. M. (BNS 8849839 and IBN- 9109851) and the NIH to R. C. B. (DC00566).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement in accordance with 18 USC Sec- tion 1734 solely to indicate this fact.

Received August 13, 1991; revised December 11, 1991.

References

Alousi, A. A., Jasper, J. R., Insel, P. A., and Motulsky, H. J. (1991). Stoichiometry of receptor-G,-adenylate cyclase interaction. FASEB J. 5, 2300-2303.

Anholt, R. R. H. (1989). Molecular physiology of olfaction. Am. J. Physiol. 257, C1043-C1054.

Anholt, R. R. H. (1991). Odor recognition and olfactory transduction: the new frontier. Chem. Senses 76, 421-427. Anholt, R. R. H., and Rivers, A. M. (1990). Olfactory transduction: cross-talk between second messenger systems. Biochemistry29, 4049-4054.

Anholt, R. R. H., Mumby, S. M., Stoffers, D.A., Cirard, P. R., Kuo, J. F., and Snyder, S. H. (1987). Transduction proteins of olfactory receptor cells: identification of guanine nucleotide binding proteins and protein kinase C. Biochemistry 26, 788-795. Asanuma, N., and Nomura, H. (1991). Cytochemical localization of adenylate cyclase activity in rat olfactory cells. Histochem. J. 23, 83-90.

Bakalyar, H. A., and Reed, R. R. (1990). Identification of a special- ized adenylyl cyclase that may mediate odorant detection. Sci- ence 250, 1403-1406. Bakalyar, H. A., and Reed, R. R. (1991). The second messenger cascade in olfactory receptor neurons. Curr. Opin. Neurobiol. 7, 204-208, 284-285.

Bendayan, M., and Garzon, S. (1988). Protein G-gold complex: comparative evaluation with protein A-gold for high-resolution immunocytochemistry. J. Histochem. Cytochem. 36, 597-607.

Besharse, J. C., and Horst, C. J. (1990). The photoreceptor con- necting cilium. A model for the transition zone. In Ciliary and Flagellar Membranes, R. A. Bloodgood, ed. (New York: Plenum Publishing Corp.), pp. 389-417.

Boyle,A. G., Park, Y. S., Huque,T., and Bruch, R. C. (1987). Proper- ties of phospholipase C in isolated olfactory cilia from the channel catfish (Ictalurus punctatus). Comp. Biochem. Physiol. &JB, 767-775.

Bradford, M. M. (1976). A rapid and sensitive method for quantita- tion of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem. 72, 248-254.

Bruch, R. C., and Carr, V. M. (1991). Rat olfactory neurons express a 200 kDa neurofilament. Brain Res. 550, 133-136.

Bruch, R. C., and Gold, G. H. (1990). G-protein-mediated signal- ling in olfaction. In G-Proteins as Mediators, M. D. Houslay and G. Milligan, eds. (New York: John Wiley & Sons), pp. 113-124.

Bruch, R. C., and Kalinoski, D. L. (1987). Interaction of GTP- binding regulatory proteins with chemosensory receptors. J. Biol. Chem. 262, 2401-2404.

Bruch, R. C., and Teeter, J. H. (1990). Cyclic AMP links amino acid chemoreceptors to ion channels in olfactory cilia. Chem. Senses 75, 419-430.

Bruch, R. C., Kalinoski, D. L., and Kare, M. R. (1988). Biochemistry of vertebrate olfaction and taste. Annu. Rev. Nutr. 8, 21-42.

Buck, L., and Axel, R. (1991). A novel multigene family may en- code odorant receptors: a molecular basis for odor recognition. Cell 65, 175-187.

Burton, P. R. (1992). Ultrastructural studies of the microtubules and microtubule-organizing centers of the vertebrate olfactory neuron. Microsc. Res. Techn., in press. Carlemalm, E., and Villiger, W. (1989). Low temperature embedding. In Techniques in Immunocytochemistry. Vol. 4, G. R. Bul- lock and P. Petrusz, eds. (London: Academic Press, Inc.), pp. 29- 45.

Carr, V. M., Farbman, A. I., Lidow, M. S., Coletti, L. M., Hemp- stead, J. L., and Morgan, J. I. (1989). Developmental expression of reactivity to monoclonal antibodies generated against olfactory epithelia. 1. Neurosci. 9, 1179-1198.

Carr, V. M., Farbman, A. I., Coletti, L. M., and Morgan, J. I. (1991). Identification of a new nonneuronal cell type in rat olfactory epithelium. Neuroscience 45, 433-449.

Chen,Z., Pace, U., Heldman, J., Shapira,A., and Lancet, D. (1986). Isolated frog olfactory cilia: a preparation of dendritic membranes from chemosensory neurons. J. Neurosci. 6, 2146-2154.

Dhallan, R. S., Yau, K.-W., Schrader, K. A., and Reed, R. R. (1990). Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurones. Nature347,184- 187.

Doty, R. L., Kreiss, D. S., and Frye, R. E. (1990). Human odor intensity perception: correlation with frog epithelial adenylate cyclase activity and transepithelial voltage response. Brain Res. 527, 130-134. Duncan, C. J. (1977). A note on the evolution of the transducer mechanism of the vertebrate retinal rod. Experientia 33, 1310. Edelmann, L. (1988). The cell water problem posed by electron microscopic studies of ion binding in muscle. Scanning Microsc. 2, 851-865. Farbman, A. I., and Margolis, F. L. (1980). Olfactory marker protein during ontogeny: immunohistochemical localization. Dev. Biol. 74, 205-215.

Firestein, S., Darrow, B., and Shepherd, C. M. (1991). Activation of the sensory current in salamander olfactory receptor neurons depends on a G protein-mediated CAMP second messenger system. Neuron 6, 825-835. Frings, S., and Lindemann, B. (1991). Current recording from

NWNXl 452

sensory cells of olfactory receptor ceils in situ. I. The neuronal response to cyclic nucleotides. j. Cen. Physiol. 97, 1-16.

Cetchell, T. V., Heck, G. IL., DeSimone, j. A., and Price, S. (1980). The location of olfactory receptor sites. Inferences from latency measurements. Biophys. J. 29, 397-412. Cilman, A. G. (1987). C proteins: transducers of receptor- generated signals. Annu. Rev. Biochem. 56, 615-649.

Cold, G. H., and Nakamura, T. (1987). Cyclic nucleotide-gated conductances: a new class of ion channels mediates visual and olfactory transduction. Trends Pharmacol. Sci. 8, 312-316.

Heuser, j. E., Reese,T. S., Dennis, M. J., Jan, Y., Jan, L., and Evans, L. (1979). Synaptic vesicle exocytosis captured by quick freezing and correlated with quanta1 transmitter release. J. Cell Biol. 81, 275-300.

Jones, D. T. (1990). Distribution of the stimulatory GTP-binding proteins, C, and G,I,, within olfactory neuroepithelium. Chem. Senses 15, 333-340. Jones, D. T., and Reed, R. R. (1989). G,,,: an olfactory neuron specific C protein involved in odorant signal transduction. Sci- ence 244, 790-795.

Jones, D. T., Masters, S. B., Bourne, H. R., and Reed, R. R. (1990). Biochemical characterization of three stimulatory CTP-binding proteins. The large and small forms of C, and the olfactory- specific G-protein, &I!. J. Biol. Chem. 265, 2671-2676.

Kaupp, U. B. (1991). The cyclic nucleotide-gated channels of vertebrate photoreceptors and olfactory epithelium. Trends Neu- rosci. 74, 150-157.

Kellenberger, E. (1991). The potential of cryofixation and freeze- substitution: observations and theoretical considerations. J. Mi- crosc. 167, X33-203. Kinnamon, S. C., and Cetchell, T. V. (1991). Sensory transduction in olfactory receptor neurons and gustatory receptor cells. In Smell and Taste in Health and Disease, T. V. Cetchell, R. L. Doty, L.M. Bartoshuk, and J. B. Snow, eds. (New York: Raven Press), pp. 145-172.

Kleene, S. J., and Cesteland, R. C. (1991). Transmembrane currents in frog olfactory cilia. J. Membr. Biol. 720, 75-81.

Kolesnikov, S. S., Zhainararov, A. B., and Kosolapov,A. V. (1990). Cyclic nucleotide-activated channels in the frog olfactory receptor plasma membrane. FEBS Lett. 266, 96-98.

Krupinski, J., Coussen, F., Bakalyar, H. A., Tang, W.-J., Feinstein, P. G., Orth, K., Slaughter, C., Reed, R. R., and Gilman, A. G. (1989). Adenylyl cyclase amino acid sequence: possible channel- or transporter-like structure. Science 244, 1558-1564. Kurahashi,T. (199O).The response induced by intracellular cyclic AMP in isolated olfactory receptor cells of the newt. J. Physiol. 430, 355-371.

Kurahashi,T., and Kaneko,A. (1991). High density of CAMP-gated channels at the ciliary membrane in the olfactory receptor cell. NeuroReport 2, 5-8. Kurihara, K., and Koyama, N. (1972). High activity of adenylyl cyclase in olfactory and gustatory organs. Biochem. Biophys. Res. Commun. 48, 30-34.

Labarca, P., and Bacigalupo, J. (1988). Ion channels from chemosensory olfactory neurons. J. Bioenerg. Biomembr. 20, 551-569.

Lancet, D. (1988). Molecular components of olfactory reception and transduction. In Molecular Neurobiology of the Olfactory System. Molecular, Membranous, and Cytological Studies, F. L. Margolis and T. V. Getchell, eds. (New York: Plenum Publishing Corp.), pp. 25-50. Lancet, D., Shafir, i,, Pace, U., and Lazard, D. (1989). Receptor- activated chemosensory cascades. In Chemical Senses. Vol. 1. Receptor Events and Transduction in Olfaction and Taste, J. C. Brand, J. H. Teeter, R. H. Cagan, and M. R. Kare, eds. (New York: Marcel Dekker), pp. 263-281. Lazard, D., Barak, Y., and Lancet, D. (1989). Bovine olfactory cilia preparation: thiol-modulated odorant-sensitive adenylyl cyclase. Biochim. Biophys. Acta 7073, 68-72.

Leunissen, J. L. M. (1990). Background suppression using Aurion BSA-C and/or Tween 20’. Aurion Newsletter i, October (Wagen- ingen, The Netherlands).

Lidow, M. S., and Menco, 8. P. M. (1984). Observations on axonemes and membranes of olfactory and respiratory cilia in frogs and rats using tannic acid-supplemented fixation and photo- graphic rotation. J. Ultrastruct. Res. 86, 18-30. Lowe, G., and Gold, G. H. (1991). The spatial distributions of odorant sensitivity and odorant-induced currents in salamander olfactory receptor cells. J. Physiol. 442, 147-168.

Lowe, C., Nakamura, T., and Gold, G. H. (1989). Adenylate cyciase mediates olfactory transduction for a wide variety of odorants. Proc. Natl. Acad. Sci. USA 86, 5641-5645.

Ludwig, J., Margaiit, T., Eismann, E., Lancet, D., and Kaupp, U. E. (1990). Primary structure of CAMP-gated channel from bovine olfactory epithelium. FEBS Lett. 270, 24-29.

Mania-Farnell, B., and Farbman, A. I. (1990). Immunohistochemi- cal localization of guanine nucleotide-binding proteins in rat olfactory epithelium during development. Dev. Brain Res. 51, 103-112. Margolis, F. L. (1972). A brain protein unique to the olfactory bulb. Proc. Natl. Acad. Sci. USA 69, 1221-1224. Margolis, F. L. (1982). Olfactory marker protein. Stand. J. Immu- nol. 75 (SuppI.), 9, 181-199. Memo, B. P. M. (198Oa). Qualiiative and quantitative freeze-fracture studies on olfactory and nasal respiratory structures of frog, ox, rat, and dog. I. A general survey. Cell Tissue Res. 207, 183- 209.

Menco, B. P. M. (198Ob). Qualitative and quantitative freeze- fracture studies on epithelial surfaces of frog, ox, rat, and dog. II. Cell apices, cilia, and microvilli. Cell Tissue Res. 277, 5-30.

Menco, B. P.M. (1984). Ciliated and microvillous structures of rat olfactory and nasal respiratory epithelia. A study using ultra- rapid cryo-fixation followed by freeze-substitution or freeze- etching. Cell Tissue Res. 235, 225-241.

Menco, B. P. M. (1986). A survey of ultra-rapid cryofixation methods with particular emphasis on applications to freeze-fractur- ing, freeze-etching, and freeze-substitution. J. Electron Microsc. Techn. 4, 177.-240.

Memo, B. P.M. (1989a). Olfactory and nasal respiratoryepithelia, and foliate taste buds visualized with rapid-freeze freeze-substitution and Lowicryl KIIM embedding. Ultrastructural and initial cytochemical studies. Scanning Microsc. 3, 257-272.

Memo, B. P. M. (1989b). Electron microscopic demonstration of olfactory-marker protein with protein G-gold in freeze-substituted, Lowicryl KIIM-embedded rat olfactory receptor cells. Cell Tissue Res. 256, 275-281.

Menco, B. P. M. (1992a). Ultrastructural studies on membrane, cytoskeletal, mucous and protectivecompartments in olfaction. Microsc. Res. Techn., in press.

Menco, B. P. M. (1992b). Lectins bind differentially to cilia and microvilli of major and minor cell populations in olfactory and nasal respiratory epithelia. Microsc. Res. Techn., in press.

Menevse, A., Dodd, G., and Poynder, T. M. (1977). Evidence for the specific involvement of cyclic AMP in the olfactory transduction cascade. Biochem. Biophys. Res. Commun. 77, 671-677. Minor, A. V., and Sakina, N. L. (1973). The role of cyclic adenosine-3’-5’-monophosphate in olfactory reception. Neirofiz- iologia 5, 415-422. Nakamura, T., and Gold, C. H. (1987). A cyclic-nucleotide gated conductance in olfactory receptor cilia. Nature 325, 442-444.

Novoselov, V. I., Krapivinskaya, L. D., Krapivinsky, G. B., and Fesenko, E. E. (1988). GTP-binding protein associated with amino acid binding proteins from olfactory epithelium of the skate, Dasyatis pastinaca. FEBS Lett. 234, 471-474.

O’Connell, R. J., Costanzo, R. M., and Hildebrandt, J. D. (1990). Adenylyl cyclase activation and electrophysiological responses elicited in male hamster olfactory receptor neurons by compo-


nents of female pheromones. Chem. Senses 75, 725-739. Pace, U., and Lancet, D. (1986). Olfactory GTP-binding protein: signal-transducing polypeptide of vertebrate chemosensory neurons. Proc. Natl. Acad. Sci. USA 83, 4947-4951.

Pace, U., Hanski, E., Salomon, Y., and Lancet, D. (1985). Odorant- sensitive adenylate cyclase may mediate olfactory reception. Na- ture 316, 255-258.

Parfenova, E. V., and Etingof, R. N. (1988). The participation of GTP-binding proteins in olfactory reception in vertebrates. Bio- chemistry (USSR) 53. 435-443

Pfeuffer, E., Mollner, S., Lancet, D., and Pfeuffer, T. (1989). Olfac- tory adenylyl cyclase. Identification and purification of a novel enzyme form. J. Biol. Chem. 264, 18803-18807.

Phillips, G. W., and Bridgman, P. C. (1991). lmmunoelectron microscopy of acetylcholine receptors and 43 kD protein after rapid freezing, freeze-substitution, and low-temperature embedding in Lowicryl KIIM. J. Histochem. Cytochem. 39, 625-634.

Phillips, T. E., and Boyne, A. F. (1984). Liquid nitrogen-bound quick freezing: experiences with bounce-free delivery of cholin- ergic nerve terminals to a metal surface. J. Electron Microsc. Techn. 7,9-29.

Pyatkina, G. A., and Dmitrieva, T. M. (1990). Cytochemical demonstration of adenylate and guanylate cyclases in olfactory mu- cosa of the grayling Thymallus arcticus baikalensis; the effect of industrial pollution. J. Evol. Biochem. Physiol. (USSR) 26, 189- 193.

Reed, R. R. (1990). How does the nose know? Cell 60, l-2.

Restrepo, D., Miyamoto, T., Bryant, B. P., and Teeter, J. H. (1990). Odor stimuli trigger influx of calcium into olfactory neurons of the channel catfish. Science 249, 1166-1168.

Ronnett, G. V., Parfitt, D. J., Hester, L. D., and Snyder, S. H. (1991). Odorant-sensitive adenylyl-cyclase: rapid, potent activation and desentization in primary olfactory neuronal cultures. Proc. Natl. Acad. Sci. USA 88, 2366-2369.

Roof, D. J., and Heth, C. A. (1988). Expression of transducin in retinal rod photoreceptor outer segments. Science 247,845-846.

Roof, D. J., and Heuser, J. E. (1982). Surfacesof rod photoreceptor disk membranes: integral membrane components. J. Cell Biol. 95, 487-500.

Seifert, K. (1970). Die Ultrastruktur des Riechepithels beim Mak- rosmatiker. Eine elektronenmikroskopische Untersuchung. In Normale und pathologische Anatomie. Heft 21, W. Bargmann, and W. Doerr, eds. (Stuttgart: Georg Thieme Verlag).

Shirley, S. G., and Persaud, K. C. (1990). The biochemistry of vertebrate olfaction and taste. Seminars Neurosci. 2, 59-68. Shirley, S. G., Robinson, C. J., and Dodd, G. H. (1986). Olfactory adenylate cyclase of the rat. Stimulation by odorants and inhibition by Ca*+. Biochem. J. 240, 605-607.

Simonds, W. F., Goldsmith, P. K., Codina, J., Unson C. G., and Spiegel, A. M. (1989). Gil mediates az-adrenergic inhibition of adenylyl cyclase in platelet membranes: in situ identification with G. C-terminal antibodies. Proc. Natl. Acad. Sci. USA 86, 7809-7813.

Sklar, P. B., Anholt, R. R. H., and Snyder, S. H. (1986). The odorant-sensitive adenylate cyclase of olfactory receptor cells. J. Biol. Chem. 267, 1553815543. Snyder, S. H., Sklar, P. B., and Pevsner, J. (1988). Molecular mech- anisms of olfaction. J. Biol. Chem. 263, 13971-13974. Spiegel,A. M., Backlund, P. S., Jr., Butrynski, J. E., Zones, T. L. Z., and Simonds, W. F. (1991). The G protein connection: molecular basis of membrane association. Trends Neurosci. 76, 338-341. Suzuki, N. (1989). Voltage- and cyclic nucleotide-gated currents in isolated olfactory receptor cells. In Chemical Senses. Vol. 1. Receptor Events and Transduction in Olfaction and Taste, J. G. Brand, J. H. Teeter, R. H. Cagan, and M. R. Kare, eds. (New York: Marcel Dekker), pp. 469-493. Trotier, D., Rosin, J.-F., and MacLeod, P. (1989). Channel activities in in vivo and isolated olfactory receptor cells. In Chemical

Senses. Vol. 1. Receptor Events and Transduction in Olfaction and Taste, J. G. Brand, J. H. Teeter, R. H. Cagan and M. R. Kare, eds. (New York: Marcel Dekker), pp. 427-448.

van Veen, T., ostholm, T., Gierschik, P., Spiegel, A., Somers, H. W., Korf, H.W.,and Klein, D. C. (1986).a-Transducin immunoreactivity in retinae and sensory pineal organs of adult vertebrates. Proc. Natl. Acad. Sci. USA 83, 912-916.

Vinnikov, Y. A. (1982). Evolution of Receptor Cells. Cytological, Membranous and Molecular Levels (Berlin: Springer-Verlag).

Vodyanoy, V. (1991). Cyclic AMP-sensitive ion channels in olfactory receptor cells. Chem. Senses 76, 175-180.

Ziegelberger, C.,van den Berg, M. J., Kaissling, K.-E., Klumpp, S., and Schultz, J. E. (1990). Cyclic GMP levels and guanylate cyclase activity in pheromone-sensitive antennae of the silkmoths An- thereaea polyphemus and Bombyx mori. J. Neurosci. 70, 1217- 1225.

Note Added in Proof

In a recent report, Drinnen et al. showed that C,lt. also occurs in neurons other than olfactory receptor cells (Drinnan, S. L., Hope, B. T., Snutch, T. P., and Vincent, S. R. [1991]. G,lf in basal ganglia. Mol. Cell. Neurosci. 2, 66-70). This implies that Co,,,, is not an olfactory neuron-specific C protein. However, this finding does not negate our observation, that within the apical parts of olfactory receptor cells G,,I~, is present mainly in those regions that most likely first encounter odorous substances, namely, the distal parts of the olfactory cilia.

Date post:	03-Jan-2017
Category:	Documents
Upload:	waleed
View:	219 times
Download:	3 times

Ultrastructural localization of olfactory transduction components: the G protein subunit Golfα and...

Documents