Manuscript 814 Words 8.471 1 Received 13 December 2010 2 3 4 5 6 7 Are olfactory receptors really olfactive? 8 9 Franco Giorgi*, Roberto Maggio** and Luis Emilio Bruni*** 10 * Corresponding author 11 Department of Neuroscience, University of Pisa 12 [email protected]13 **Department of Experimental Medicine, University of L’Aquila and 14 ***Department of Media Technology and Engineering Science, Aalborg University, Denmark 15 16 17 Abstract 18 19 Any living organism interacts with and responds specifically to environmental molecules by 20 expressing specific olfactory receptors. This specificity will be first examined in causal terms with particular 21 emphasis on the mechanisms controlling olfactory gene expression, cell-to-cell interactions and odor- 22 decoding processes. However, this type of explanation does not entirely justify the role olfactory receptors 23 have played during evolution, since they are also expressed ectopically in different organs and/or tissues. 24 Homologous olfactory genes have in fact been found in such diverse cells and/or organs as spermatozoa, 25 testis and kidney where they are assumed to act as chemotactic sensors or renin modulators. To justify their 26 functional diversity, homologous olfactory receptors are assumed to share the same basic role: that of 27 conferring a self-identity to cells or tissues under varying environmental conditions. By adopting this 28 standpoint, the functional attribution as olfactory or chemotactic sensors to these receptors should not be 29 seen either as a cause conditioning receptor gene expression, or as a final effect resulting from genetically 30 predetermined programs, but as a direct consequence of the environmental conditions olfactory receptor 31 genes have explored during evolution. The association of odorant patterns with specific environmental or 32 contextual situations makes their relationship semiotically triadic, due to the emergence of an interpretant 33 capable of perceiving odorants as meaningful signs out of a noisy background. This perspective highlights 34 the importance of odorant-receptor relationships as respect to the properties of the interacting partners. It is 35 our contention that only when taken together can these different explanatory strategies provide a realistic 36 account of how olfactory receptor genes have been structurally and functionally modified during evolution. 37 38 39 Keywords 40 41 Olfaction, chemotactic, pheromonal, trichromasy, causal and functional explanations 42 43 44 Introduction 45 46 The question of whether olfactory receptors are really olfactive may sound rather meaningless, as odor 47 discrimination is usually considered their sole activity. However, the observation that they are also expressed 48 in such diverse tissues as testis (Fukuda et al., 2004) and renal distal nephrons (Pluznick et al., 2009), besides 49 the olfactory epithelium (Vassar et al., 1993), raises the question of how their structural communality can 50 actually generate such an astonishing functional diversity. Olfactory receptors constitute the largest gene 51 family amongst mammalian G-protein-coupled receptors (Fleischer et al., 2009) and exhibit such prominent 52 features as a high intraspecific variability, remarkable discriminative repertoires of environmental molecules 53 and numerous pseudogenes (Shi and Zhang, 2009). It is through the realization of these features that animals 54 can actually search for food, accomplish mating and care for their offspring, and occasionally flee to avoid 55
Transcript
Manuscript 814 Words 8.471 1 Received 13 December 2010 2 3 4 5 6 7
Are olfactory receptors really olfactive? 8
9
Franco Giorgi*, Roberto Maggio** and Luis Emilio Bruni*** 10 * Corresponding author 11
Department of Neuroscience, University of Pisa 12 [email protected] 13
**Department of Experimental Medicine, University of L’Aquila and 14 ***Department of Media Technology and Engineering Science, Aalborg University, Denmark 15
16 17
Abstract 18 19
Any living organism interacts with and responds specifically to environmental molecules by 20 expressing specific olfactory receptors. This specificity will be first examined in causal terms with particular 21 emphasis on the mechanisms controlling olfactory gene expression, cell-to-cell interactions and odor-22 decoding processes. However, this type of explanation does not entirely justify the role olfactory receptors 23 have played during evolution, since they are also expressed ectopically in different organs and/or tissues. 24 Homologous olfactory genes have in fact been found in such diverse cells and/or organs as spermatozoa, 25 testis and kidney where they are assumed to act as chemotactic sensors or renin modulators. To justify their 26 functional diversity, homologous olfactory receptors are assumed to share the same basic role: that of 27 conferring a self-identity to cells or tissues under varying environmental conditions. By adopting this 28 standpoint, the functional attribution as olfactory or chemotactic sensors to these receptors should not be 29 seen either as a cause conditioning receptor gene expression, or as a final effect resulting from genetically 30 predetermined programs, but as a direct consequence of the environmental conditions olfactory receptor 31 genes have explored during evolution. The association of odorant patterns with specific environmental or 32 contextual situations makes their relationship semiotically triadic, due to the emergence of an interpretant 33 capable of perceiving odorants as meaningful signs out of a noisy background. This perspective highlights 34 the importance of odorant-receptor relationships as respect to the properties of the interacting partners. It is 35 our contention that only when taken together can these different explanatory strategies provide a realistic 36 account of how olfactory receptor genes have been structurally and functionally modified during evolution. 37 38 39 Keywords 40 41 Olfaction, chemotactic, pheromonal, trichromasy, causal and functional explanations 42 43 44 Introduction 45 46 The question of whether olfactory receptors are really olfactive may sound rather meaningless, as odor 47 discrimination is usually considered their sole activity. However, the observation that they are also expressed 48 in such diverse tissues as testis (Fukuda et al., 2004) and renal distal nephrons (Pluznick et al., 2009), besides 49 the olfactory epithelium (Vassar et al., 1993), raises the question of how their structural communality can 50 actually generate such an astonishing functional diversity. Olfactory receptors constitute the largest gene 51 family amongst mammalian G-protein-coupled receptors (Fleischer et al., 2009) and exhibit such prominent 52 features as a high intraspecific variability, remarkable discriminative repertoires of environmental molecules 53 and numerous pseudogenes (Shi and Zhang, 2009). It is through the realization of these features that animals 54 can actually search for food, accomplish mating and care for their offspring, and occasionally flee to avoid 55
danger. In these circumstances of ever changing odor combinations, it is crucial for the animal’s survival to 56 verify whether newly encountered environmental signals match anticipated expectations. In the end, these 57 recurrent olfactive interactions allow the animal to establish a comprehensive image of the environment – its 58 Umwelt – and to gain access to acquired or newly learned olfactive patterns (Wilson and Leon, 1988; 59 Sullivan and Wilson, 2003). 60
To account for this highly discriminative power of olfactory receptors, along with their ectopically 61 differentiated distribution, several explanations are possible. In this paper, we will first show how this power 62 could be accounted for by a number of molecular mechanism explaining how odorants interact with their 63 respective olfactory receptors, within a certain binding threshold. Even though this mechanistic causal 64 explanation may ultimately justify several of the cell and molecular specificities realized by the olfactory 65 epithelium, it does not entirely explain how and why olfactory receptors have evolved into such a variety of 66 roles and cell types (Dreyer, 1998; Derby and Steullet, 2001). In a second approach, we will be arguing that 67 olfactory receptors could be explained in functional terms, i.e., justified for the function they guarantee, 68 whenever expressed in competent organisms. This type of approach is inevitably teleological (Buller, 1999) 69 and bound to subsume their usefulness to the temporal asymmetry of their cause-effect relationships, i.e., 70 their functional accomplishment could be understood as if planned ahead and due to some kind of goal-71 directed development (Nagel, 1977). Consequently, it would leave unanswered the question how certain 72 ligand-receptor interactions could have been selected before cells were fully equipped with functional 73 signaling pathways (Soyer and Bonhoeffer, 2006). 74
Causal and functional analyses are both reductionistic approaches that attempt to explain dyadically 75 different types of receptor-ligand interactions as if they are to single-cause to single-effect relationships. 76 However, biological relations should also be considered as context-dependent processes, and therefore their 77 tissue and organ expression can be justified in terms of the semiotic context they are part of (Emmeche, 78 2000). What could emerge as potentially useful in a certain context, could turn out to be genuinely 79 meaningful in another, if recognized as a sign supporting some mechanisms for the maintenance of specific 80 relationships. But this would require the organism and/or the tissue expressing these receptor specificities to 81 behave as an evolved complex system interacting qua agent with the sign-objects of a triadic world 82 (Atmanspacher, 2005). Accordingly, to explain the ligand specificity and the ectopic diversity of the 83 olfactory receptors would imply considering the organism as an emerging interpretant capable of rearranging 84 heterarchically its internal dynamics in response to any meaningful combination of environmental odors 85 (Bruni, 2008, Hoffmeyer, 2008). Heterarchy in this context is meant to emphasize the notion that categories 86 of different logical levels may relate to one another as a network of emerging processes, rather than being 87 simply vertically subordinated, as in hierarchical relations. 88
In this paper, the appropriateness of the causal, functional and biosemiotic approaches will be critically 89 evaluated and their respective explanatory powers considered as complementary to one another, rather than 90 mutually exclusive. 91
92 93
Causal analysis 94 95 To interpret natural phenomena in causal terms entails deducing certain regularities from general laws 96 (Nagel, 1961). For these regularities to persist, some kind of energy or information transfer has to occur 97 between causally linked events (Salmon, 1984). Under these conditions, the regularity of their occurrence 98 may be attributed to some kind of governed relationship, such that antecedents are considered causes 99 determining the effects that follow. In this section, olfactory receptors and their respective odorants will be 100 examined following this theoretical framework. The most relevant experiments accounting for their role as 101 chemo-sensors of exogenous odorants will be analyzed with a particular emphasis on the mechanisms 102 controlling their conformational stability and activation states. Our aim is to account for the mechanisms by 103 which olfactory receptors interact with structurally different molecules, how they respond to their signals 104 and, eventually, how they elaborate the environmental information gained through these interactions. 105
Any living organism interacts with and responds specifically to environmental molecules. This depends 106 on their ability to detect and discriminate between a vast variety of volatile chemicals. In vertebrates, this 107 discriminative power is exerted by two distinct systems, the olfactory epithelium (OE) and the vomero-nasal 108 organ (VNO), both of which are comprised of sensory neurons expressing different types of receptors. While 109 the olfactory epithelium expresses only olfactory receptors (ORs) specific for environmental odorants, the 110 vomero-nasal organ expresses two distinct receptor types, VR1 and VR2, each specific for pheromone 111
detection (Ji et al., 2009). Both OR and VNO receptors are G-protein-couple receptors (GPCR) encoded by 112 the largest multigene family in vertebrates (Buck and Axel, 1991). The mechanism by which each of these 113 receptors interacts with specific environmental odorants has been the subject of numerous investigations. At 114 present, the available evidence indicates that olfactory receptors are characterized by the expression of 115 several hypervariable trans-membrane domains with high binding affinity for aliphatic or hydrophobic 116 hydrocarbons chains (Abbafy et al., 2007). An environmental odorant may bind specifically to its respective 117 receptor only if it expresses a conformational structure comprised of a number of rotable carbon bonds. At 118 the same time, receptors have to have a number of adaptable binding sites within their trans-membrane 119 regions such as to interact with each other and with the incoming ligand (Peterlin et al., 2008). This type of 120 ligand-receptor interaction leads inevitably to a condition in which one receptor may recognize different 121 odorants, along with the possibility for an odorant to be recognized by multiple olfactory receptors. Both 122 these conditions allow the organism to acquire certain expressive qualities and consequently perceive the 123 environment as a meaningful Umwelt. 124
It is also possible for certain olfactive receptors to interact with a combination of different odorants. 125 Needless to say that this state of affairs may ultimately result in combinatorial codes, giving rise to the 126 recognition of new odorant patterns (Dreyer, 1998; Galizia and Menzel, 2000; Stensmyr et al., 2003), and, as 127 will be clarified later on in this paper, this will eventually lead to a categorical sensing of contextual 128 conditions and to the establishment of a digital-analogical consensus (DAC) (Bruni, 2008). 129
Interestingly, every sensory neuron in the olfactory epithelium expresses only one type of receptor 130 (Malnic et al., 1999), while neurons expressing different receptors are randomly dispersed within the 131 epithelium itself (Touhara et al., 1999). However, sensory olfactory neurons expressing the same type of 132 receptor are regularly clustered in the glomerular units of the olfactory bulb (Chen and Shepherd, 2005). 133 Besides this one- receptor-to-one-cell relationship, the olfactory epithelium is also characterized by multiple 134 axonal projections, whereby stimuli perceived by the olfactory neurons that express similar receptors are 135 redundantly targeted to the same glomeruli. This anatomical link is thought to accomplish a significant 136 signal-to-noise amplification for those environmental stimuli perceived as particularly relevant for the 137 organism’s survival. In the end, information coming from the sensory neurons of the olfactory epithelium 138 project into the basal telencephalon, where they may be partly combined with pheromonal stimuli coming 139 from the vomeronasal organ (Martinez-Marcos, 2009). 140
Taking this brief description as a realistic account of the olfactory system in vertebrates, there are a 141 number of important questions that a mechanistic analysis of the sense of olfaction should help us 142 understand. For instance, one may ask: (1) what kind of cell response a specific odorant induces in the 143 targeted olfactory neuron? (2) How is the one receptor - one neuron ratio accomplished during development? 144 (3) How are neural projections targeted specifically to the same type of glomeruli? (4) And how is it possible 145 for a limited number of receptors to recognize a wide variety of environmental molecules? Let us now see 146 what kind of experimental evidence provides realistic answers to the above mechanistic questions. 147
First, the nature of the cell response elicited by olfactory neurons is not only related to the odorant’s 148 chemical nature, but also to the odor map provided by bulb glomeruli and their chemoreception decoding 149 capability (Hildebrand, 1995). Odorants must bind to the olfactory receptor with a binding energy above a 150 minimal threshold, as otherwise there would be no activation (Hummel et al. 2005). This guarantees new 151 combinatorial roles for receptors differing in their activating threshold and for odorants interacting with 152 different binding energy. Above this minimal threshold, odorant stimuli are perceived through the activation 153 of a cAMP-based transducing system, via interaction with G proteins (Firestein et al., 1991). This is known 154 to take place along the distal dendrites of every olfactory neuron. However, the observation that olfactory 155 perception is not completely abolished in mice with a defective cAMP transduction pathway, suggests that 156 olfactive signaling could also include some transient receptor potential channels downstream from the 157 phospholipase C pathway (Lin et al., 2007). In either case, the final effect of this second messenger 158
activation is mediated by modulation of the resting membrane conductance. Following alteration of their Cl_ 159
and K+ membrane conductance, olfactory neurons respond by generating action potentials in their terminal 160 cilia and eventually transfer their firing signals to the olfactory bulb (Dubin and Dionne, 1994). In 161 conclusion, a well known physiological mechanism, the abrupt change in membrane conductance as 162 triggered by GPCR activation, is also operating in specific olfactory neurons, and this is sufficient to account 163 mechanistically for the causal link connecting odor binding to cell responses. Recent evidence that makes the 164 odor activation mechanism more complex than expected is the finding that some olfactory receptors are 165 made functional if, and only if, co-expressed with adrenergic receptors along the plasma membrane of 166 olfactory neurons (Hague et al., 2004). The fact that oligomeric complexes are to be formed prior to receptor 167
activation remind us of the importance of oligomerization, as a process mediating protein folding and 168 receptor selection along the plasma membrane of many eukaryotic cells (Giorgi et al., 2010). 169
The second question raised above deals with the determination of the one-to-one ratio between receptors 170 and olfactory neurons which, as we have seen, is fundamental for constructing an odor map in the olfactory 171 epithelium and to make it capable of discriminating the large variety of molecules in the environment. Even 172 though this is primarily a developmental rather than a physiological question, it can nevertheless be 173 approached mechanistically within reasonable experimental time scales. There are in fact many 174 developmental models that make resolution of this issue possible within convenient time scales and 175 experimentally solvable in strictly proximate causal terms. In principle, there could be two different ways of 176 realizing the one-to-one ratio: either have a pre-determinate gene program conditioning receptor expression 177 in any new born neuron, or alternatively, let each neuron have its own receptor choice based on some kind of 178 feed-back mechanism sustained by specific cell interactions. What is remarkable is that both solutions have 179 been adopted by nature. Drosophila has been shown to employ a deterministic type of mechanism, even 180 though mediated by epigenetically controlled programs, whereby olfactory receptors are expressed in a cell-181 specific manner due to cis-acting elements interacting specifically with transcriptional factors (Fuss and Ray, 182 2009). Through this mechanism, a number of positive and negative regulatory elements may actually interact 183 with one another in a sort of combinatorial code to ensure that olfactory genes are properly expressed in each 184 neuron (Ray et al., 2008). 185
On the other hand, the choice of specific olfactory receptors in vertebrate neurons is basically stochastic, 186 being dictated by an H-enhancer element trans-acting on multiple OR genes and independently of their 187 chromosomal location. Olfactory genes can thus be transcribed differently in any neuron, through the 188 specific association of the H enhancer element with every olfactory promoter via protein-to-protein 189 interaction. By virtue of this mechanism, a single H-element can actually control the activation of thousands 190 of olfactory gene promoters in each vertebrate genome. The cell specificity of this mechanism is, in 191 principle, guaranteed by the relative affinity of the H enhancer for each of the multiple different OR gene 192 promoters (Lomvardas et al., 2006). However, there has to be some additional feedback mechanism(s) 193 whereby neurons expressing specific OR receptors can be targeted to the relative glomeruli in the absence of 194 any predetermined synaptic connection. And this brings us to the third question, which is how to explain 195 mechanistically the specificity of the neural projections for the same type of glomeruli. This objective is 196 apparently achieved by controlling the temporal and spatial expression of olfactory receptors in growing 197 neural axons. It has been demonstrated that receptors act as chemotropic molecules guiding the axonal path-198 finding process in differentiating olfactory neurons. During this developmental phase, neurons express 199 specific olfactory receptors along the growth cone, and by doing so, induce the local production of cAMP 200 and the activation of Ca+ channels (Maritan et al., 2009). Olfactory receptors can thus exert a dual role in 201 neuron differentiation: on one hand, define the odor specificity expressed by each neuron, and on the other, 202 determine which specific axonal contacts should be established in bulb glomeruli. The question of how 203 neurons select their olfactory receptors has also been verified experimentally by studying how long a 204 mutated olfactory receptor can be expressed within a given neuron. Evidence demonstrates that neurons 205 expressing mutated receptors have a far greater probability than wild type neurons to switch to different 206 receptor types (Shykind et al., 2004). Thus, besides determining the neuron specificity toward specific 207 odorants, the additional role of olfactory receptors in axonal guidance adds more constraints that restrict the 208 extent of stochastic cell interactions within the olfactory epithelium. By restraining their axonal growth to 209 specific synaptic contacts, the olfactory epithelium may thus attain neuronal stability and, at the same time, 210 maintain a sufficient developmental plasticity in neuronal synaptic connections. 211
Finally, the fourth and last question raised above is certainly the most problematic with respect to being 212 resolved experimentally. Knowledge of how many receptors are expressed per neural cell, how they interact 213 with chemical odorants and how activated neurons project to the olfactory bulb may not suffice to explain 214 how odorants are perceived, decoded and eventually interpreted by the organism. The one-to-one 215 relationship between olfactory receptors and neurons provide the anatomical basis for a spatial combinatorial 216 code (Galizia et al., 1999), but this may not be sufficient to discriminate the signal-to-noise ratio for each 217 environmental odorant. The alternative procedure for decoding an olfactory stimulus is through the control of 218 the temporal patterning by which specific odorants activate different glomeruli. A synchronous volley of 219 action potentials may be decoded differently from an asynchronous volley in the olfactory epithelium 220 (Galizia and Metzel, 2000). Certain olfactory responses might be sensitive to the initial conditions of 221 receptor occupancy and G protein activation. Any behavioral response induced by these dynamical change 222 may thus be more sensible to experienced odors and perceptions, and therefore depend upon a history of 223
regressed stimulations (Broome et al., 2006). This possibility has been clearly demonstrated in the plant 224 pollinator moth Manduca sexta where the synchronized activity of the glomerular units provides the essential 225 mechanism for processing a stream of overlapping odor stimuli into a coherent percept (Riffel et al., 2009). 226
In conclusion, the causal explanation offered in this section, with its particular emphasis on olfactory gene 227 expression, on cell-to-cell interactions and odor-decoding mechanisms in the olfactory epithelium provides a 228 realistic account of how living organisms have come to be endowed of such a highly discriminative sense of 229 smell. However, in our view, this type of explanation does not entirely justified how olfactory receptor genes 230 have evolved, why have they attained such high level of tissue expression and what role have they played in 231 the establishment of meaningful relationships with the environment. 232
233 234
Functional Analysis 235 236
The opportunity to verify whether certain phenomena are deterministically linked provides us with some of 237 the most logically reliable models for understanding causation in nature. One of the main reasons why causal 238 explanation has been pursued by philosophers as the most reliable logical model to account for natural 239 phenomena is the temporal asymmetry by which causes and effects are mechanically linked (Goudge, 1958). 240 The existence of such an asymmetrical connection makes regular occurrences experimentally verifiable and, 241 above all, makes them justifiable in counterfactual forms whereby in the absence of causes even the 242 following effects should not be expected (Athanasiadou and Dirven, 1997). Thus, if deterministically 243 qualified, causes are necessary and sufficient events for the following effects to happen. For a phenomenon 244 to be fully pre-deterministic several conditions have to be satisfied. The phenomenon has to be entirely 245 dependent on initial conditions; it must be studied in separation from any environmental influence; and it has 246 to be justified in terms of object properties (Mazzocchi, 2008). However, biological phenomena are complex 247 non-linear systems that defy any deterministic approach and cannot therefore be studied in situations 248 separated from any realistic relationship with their surroundings. To account for the evolutionary and 249 developmental acquisition of newly acquainted properties biological phenomena could be more appropriately 250 studied in functional terms and therefore interpreted as if they were somehow goal-directed, i.e., oriented 251 toward the realization of specific end-products. 252
In this section we will try to examine olfactory receptors and the function they play in different species 253 and organs by taking into account the theoretical framework outlined above. We will thus consider whether it 254 would be legitimate for us to extend the olfactive function of these receptors to organs or tissues other than 255 the olfactory epithelium. We will also consider whether the function attributable to these receptors, be it 256 olfactive or chemotactic, could be reasonably qualified as a proper function or, rather be attributed to an 257 epistemically biased operation (Griffith, 1993). In the end, we should also ask whether these functional 258 attributions could be considered responsible for the receptor’s selection or, on the contrary, whether they 259 could simply be considered as the ultimate acquisition of the organism’s adaptation. 260
These questions stem directly from the observation that olfactory receptors are expressed ectopically in 261 many organs and/or tissues other than the olfactory epithelium where they were originally identified. By 262 using data collected from various mouse and human transcriptome sources, Feldmesser et al. (2006) have 263 recently demonstrated that olfactory genes are widely expressed in such diverse organs as testis, kidney and 264 spermatozoa. It should be recalled at this juncture that homologous gene expression is experimentally 265 established through base sequence comparison (Turchin and Kohane, 2002), which indicates, therefore, that 266 homologous genes are not necessarily functionally equivalent, for they may come to realize different 267 functions in different tissues. The fact that olfactory genes were first identified in the olfactory epithelium 268 has placed some kind of historical legacy on them such that any subsequent functional attribution has been 269 conditioned by this initial interpretation. For instance, the finding that the mouse olfactory gene MOR23 is 270 also expressed in the testis has been interpreted as due to its capacity to control a sex-pheromone-induced 271 behaviour (Fukuda et al., 2004). Accordingly, this mouse olfactory gene has been assumed to function in the 272 fertilization process as if it could condition the spermatozoon olfactory perception while it migrates toward 273 the egg. The presence of olfactory receptors in spermatozoa makes them potentially capable of perceiving 274 the same molecular receptive fields as if expressed in the nose. Such chemo-attractants as bourgeonal and 275 undecanal proved capable of conditioning the activation and/or inhibition of several olfactory receptors, 276 regardless of the tissue they are actually expressed (Spehr et al., 2003). More recently, olfactory receptors 277 and their associated signalling pathways were shown to mediate spermatozoa hypermotility, as induced by 278 exposure to progestin. This indicates that spermatozoa can modify their behaviour in response to specific 279
hormone(s), thus suggesting that their functional role is not an epistemically biased attribution (Tubbs and 280 Thomas, 2009). Olfactory receptors are also expressed in primordial germinal cells to control migration 281 during gonadic development (Goto et al., 2001). Even more intriguing is the finding that olfactory receptors 282 are also expressed in the kidney. Here, co-localization with adenylate cyclase and olfactory G proteins makes 283 them functionally active for smelling the filtered tubular fluid and perhaps for controlling the renin plasma 284 levels (Pluznick et al., 2009). Taken together, all this evidence indicates that olfactory receptor genes, or 285 their homologous genes, are indeed ectopically expressed in various tissues, and that any functional 286 attribution is made difficult by the historical legacy due to their first identification as members of the 287 olfactory epithelium. 288
Functional explanations are often justified in etiological terms, i.e. new functions may emerge and persist 289 during evolution because they make the organism fitter. Assuming ectopic expression of olfactory receptor 290 genes to be advantageous to the bearing organisms, then variations in gene expression would be expected to 291 correlate with the inherited receptor phenotypes. In the absence of such a correlation, any variations in gene 292 expression would have to be intended as evolutionarily neutral (Yanai et al., 2004). An extensive sequence 293 analysis has shown that olfactory genes can be classified into several clades that do not correlate 294 phylogenetically with their chromosomal location. It is assumed that such a genomic clustering might have 295 occurred during evolution through repetitive gene duplication of different chromosome regions containing 296 olfactory genes (Niimura and Nei, 2001). Lai et al. (2008) have clearly demonstrated that the human gene 297 hOR17-210 is no longer functional due to a mutated transmembrane helical domain that makes olfactory 298 receptors improperly folded. Perhaps it is through recurrent and random genomic integrations of reversely 299 transcribed mRNAs that functional olfactory genes have been reduced to 400 in humans, while maintained at 300 a level of about a thousand in mice (Niimura and Nei, 2006). Although mutated olfactory genes are 301 evolutionarily neutral and make no contribution to the receptor functionality, they are not totally removed 302 from the genome, but retained as non-functional gene relics or pseudogenes (Gilad, et al 2003). Pseudogenes 303 are estimated to be at least ~20,000 in the human genome, a figure amounting to about 70% of the entire 304 human olfactory gene repertoire (Menashe et al., 2006). 305
It has been observed that human olfactory genes are scattered on almost all chromosomes – except two, 306 the 20th and the Y chromosomes – and that they are closely linked to genes coding for the human immune 307 histo-compatibility (HLA). The close linkage between HLA and OR genes might represent a form of 308 evolutionary adaptation to guarantee the self/non-self recognition during fertilization and/or olfaction-driven 309 mating (Ziegler et al., 2002). Apparently, the so called sexually-selected sperm or sperm competition 310 hypothesis could be explained by assuming that spermatozoa are selected following some kind of HLA 311 recognition, although in principle other physiological or behavioral factors could also explain the same 312 phenomenon (Pizzari and Birkhead, 2002). 313
In our view what makes this hypothesis interesting is that the ectopic expression of these receptors could 314 finally be explained without being biased by any historically acquainted narrative. The possibility of self 315 recognition is in fact basic to any biological phenomenon and it is essential for defining any biological 316 identity. Nasal olfaction, spermatozoa chemotaxis, primordial germ cell migration and nephron sensitivity 317 could all be sharing the same essential role: i.e. to guarantee specific interaction with some otherness, be it 318 provided by cells, organisms or by the chemical composition of the external environment, and yet be capable 319 of distinguishing itself from all that is external, such as to maintain a proper identity. If this hypothesis could 320 be experimentally validated, then it could also provide an interesting example of how functions could have 321 been accomplished evolutionarily according to the logical framework outlined here. We are in fact assuming 322 the existence of such a basic function as a self-description and definition (Kull, 1998), as it may be initially 323 guaranteed by some kind of primordial receptor acting through an autocrine signaling loop. In the course of 324 evolution such a function might have been encoded in the gene memory system and eventually expressed in 325 tissue specific manners through varying gene recombination and chromosomal translocations. By adopting 326 this standpoint, the functional attribution as olfactory or chemotactic to these receptors should not be seen 327 neither as a cause conditioning the receptor expression or as the final effect resulting from a genetically 328 predetermined program. The fact that these receptors become olfactory or chemotactic is simply due to the 329 local contingency of their expression as it is actualized in conformity with the constraints imposed by the 330 hosting tissue(s) and the interactions they are bearing thereof. This way of thinking is in line with the so 331 called area code hypothesis whereby olfactory receptors could play key functional roles in cell recognition. 332 Such a role could explain the remarkable specificity exhibited during cell migration and tissue assembly in 333 developing embryos (Dreyer, 1998). 334
The central role played by olfactory receptors in defining a principle of self-identity is also demonstrated, 335 in our view, by the concomitant evolution of color vision in higher mammals and especially primates. It 336 should not be forgotten that olfaction has been gradually become less and less important as communication 337 sense, as primates have started to develop trichromatic color vision (Dulai et al., 2010). It is quite plausible 338 that olfaction might have been gradually substituted by vision, which had, in the process, taken over some of 339 olfaction’s communicatory role. As it will be discussed in the next section, the same role in this context is 340 not a functional attribution but a sort of biosemiotic causation that may justify the maintenance of the same 341 relationship in terms of meaningfulness or the emergence of a new Umwelt where more refined images or 342 experiences are supplemented or integrated (Kull et al., 2009). 343
344 345
Biosemiotic Analysis 346 347
In this section, we will try to show that, besides being causally explainable in their present ectopic and 348 multifunctional roles, olfactory receptors can also be justified following a biosemiotic perspective. By doing 349 so, we will be proposing that a biosemiotic approach is not only required to complement the causal and 350 functional explanations, but it may also help us understand how the olfactive role played by these receptors 351 has been gradually modified during evolution to sustain a meaningful relationship with the organism’s 352 umwelt. In our view, what the mechanical and functional explanations are missing is an appreciation of the 353 heterarchical nature of the relationship held by olfactory receptors with environmental odorants. Because of 354 this, we fail to understand how new levels of contextual sign interpretation might have emerged as their 355 relationships have proceeded from molecule discrimination to highly complex multimodal integration 356 processes. 357
A fundamental tenet of biosemiotics is that living organisms entertain meaningful relationships with the 358 environment by forming essential units with it (Aldinhas Ferreira, 2010). Although each of the two 359 interacting entities could in principle be accounted for by their physical structures, this description could 360 only aim at defining the properties required for sustaining the relationship. On the other hand, by defending 361 the primacy of the relationship, biosemioticians are actually subsuming the role played by the physical 362 properties to that played by the relationship itself, thus rendering the nature of the former ontologically 363 different from that of the latter. It is in this perspective that the large body of evidence available today on 364 olfaction will be analyzed here. For instance, we have already observed that olfactory and beta-adrenergic 365 receptors associate as oligomeres on the neuronal cell membrane as a necessary pre-condition to becoming 366 functionally active (Hague et al., 2004). This demonstrates that oligomerization may constitute a basic 367 molecular mechanism allowing receptors to explore new combinatory affinities in relation to the chemical 368 heterogeneity of the extracellular milieu (Szidonya et al., 2008). However, this phenomenon could be 369 interpreted as simply due to the chemical properties of the interacting molecular partners or, more profitably, 370 as an example of how relationships, once conventionally established, may condition any further modulatory 371 change of the related molecules (Giorgi et al., 2010). In this latter case, the oligomeric assembly driven by 372 natural selection may explore new possible interactions with environmental ligands and this would be 373 experienced by the bearing organism as a meaningful adaptive response for its survival and persistence. In 374 biosemiotic terms, the recognition of odorant patterns and the association of these patterns with specific 375 environmental or contextual situations make their relationship no longer dyadic. Under these conditions, the 376 ligand-receptor relationship is in fact bound to become triadic, due to the emergence of an interpretant that 377 perceives odorants as meaningful signs out of a mixed and noisy background (Peirce, CP 1541). The need to 378 maintain an odor specificity in the myriad of combinatorial possibilities offered by the environment points to 379 the existence of some kind of categorical sensing whereby categorically perceived patterns are causally 380 linked to specific environmental and contextual conditions. The emergent triadic causality comes thus to be 381 defined by the association between specific behavioural responses and the responding repertoire potentially 382 available to the perceiver. These combinatorial associations are amenable to a digital-analogical consensus 383 (DAC) logic (Bruni, 2008), rendering low synchronic thresholds susceptible of creating heterarchically 384 higher levels of complexities. In the case of olfactory receptors, this means that specific odorant molecules 385 differing in binding energy thresholds may combine synchronically and, together, represent the “odor-386 image” of a given umwelt, 387
On higher hierarchical levels, cell-to-cell relationships are also explored through developmentally 388 adaptable mechanisms and semiotic perceptual learning. It has been recently observed that olfaction is 389 influenced by newly born neurons and that adult neurogenesis is conditioned by both training and/or 390
environmental exposure (Yuan, 2010). This is a clear example illustrating how adult neurogenesis and 391 olfactory perceptual learning are reciprocally dependent on each other. While neural stem cells are to be 392 continuously integrated as periglomerular interneurons in the olfactory bulb for learning to occur, it is also 393 true that exposure to odorant enriched environments is the necessary requirement for newly born neurons to 394 survive and to become part of a functional neuronal network (Moreno et al., 2009). To use the metaphoric 395 language of Harries-Jones (2002), this reciprocal exchange may actually occur whenever bonds become 396 binds, that is to say, when conventional or adventitious encounters acquire their significance by developing 397 some kind of reciprocal dependency that make it possible for the relationship to persist. This leads us also to 398 rephrase the claim that what is actually changing during evolution are not single entities, be it genes, cells or 399 organisms, but entire multilevel networks. As these complexes change in structure and properties, the 400 relationships between their interacting entities are maintained in such a way as to guarantee the survival of 401 their related systems of correspondences (Bruni 2003, 2007). 402
The ability to maintain such a system of correspondence is essential for odorant stimuli to be perceived as 403 coherent percepts and be categorically sensed as analogue- products sorted out as combinations of many 404 digitally synchronous bits. An experienced “coherence” of these percepts will eventually result in the 405 concept of “semantic congruency” that is known to characterize every heterarchical process of coherently 406 bound and integrated perception (Bruni, 2010)(forthcoming). This entails the simultaneous packaging of 407 several exploring patterns and the inclusion of the resulting experienced differences into a responding 408 repertoire, that, for this very reason, comes to be encoded in the historical and expanding behavioural 409 tautology of any developing organism. The coherence or semantic congruency that is so experienced 410 becomes then a function of the history of such a tautology so that, through memory and learning processes, it 411 acquires the ability to trade off between innate and non-innate factors. 412
An educated smell is not only required for neuron survival, but also for the organism to sustain 413 meaningful relationships with the environment. Through the use of the olfactive sense, organisms can in fact 414 ascertain the presence external odorants, and eventually coordinate their internal states to find out what 415 environmental information they should regard as significant. This entails that no odorant can actually be 416 perceived as significant unless it is recognized as a sign within the responding repertoire of the perceiver. By 417 doing so, the organism comes ultimately to define a number of perceptual categorical primitives (Cariani, 418 2001), and build up some kind of hierarchical categorical perception that gradually leads from low sensing 419 levels to more sophisticated percepts, categories and “logical products”, all together making a sort of referent 420 smell-scape. At this organismic level, the question that may pertain for biosemiotics is whether olfaction can 421 be regarded as a distinct and separate function or if it may instead be part of a larger perceptual experience. 422 The issue is commonly referred to as the binding problem, whereby any perceptual categorization needs to 423 be contextualized and bound to other cognitive concepts for the organism to be able to entertain meaningful 424 relationships with the environments (Harvey and Sanchez-Vives, 2005). A clear example of how organisms 425 may actually interact meaningfully with the environment is through the perception of conspecific or 426 heterospecific pheromones. This has been made possible by the development of two anatomically distinct 427 chemosensory neurons: the olfactory epithelium and the vomeronasal organ (Keverne, 2002). It is of great 428 interest to observe that any major change occurred during evolution in the morphology and function of these 429 sensory organs has also affected organismic pheromonal communication in a manner that is functionally 430 correlated with the transition from aquatic to dry land environments. However, in spite of changing their 431 chemical properties from solubility to volatility during this transition, pheromones have maintained the same 432 semiochemical significance (Swaney and Keverne, 2009). And this might have been achieved by imposing a 433 high evolutionary pressure on maintaining the same relationship in any of the new ecological niches 434 explored. Once again, by proposing a biosemiotic type of explanation even in this context, we are actually 435 arguing that all these processes – pheromonal perception, organismic communication and environmental 436 shifts - are not only mechanistically and causally linked, but that they are also semiotically connected to 437 guarantee a meaningful organismic relationship with the environment. 438
That pheromones might have been primarily acting as semiochemical perceivers – qua sign interpretants - 439 rather than simple chemical sensors is also demonstrated by the fact that, with the evolution of trichromacy 440 and the expansion of the cortex in higher primates, the role played by pheromonal communication has 441 gradually diminished in importance as a modulator of the sexual behavior (Rouquier and Giorgi, 2007; Hurst 442 and Beynon, 2010). It is as if the evolution of trichromacy by duplication of two separate X-linked opsin 443 genes (Dulai et al., 2010) has made it possible for color vision to substitute for the pheromonal and olfactory 444 perceptions in the same type of cognitive relationship. Through this functional replacement, higher 445 organisms might have developed new and more resolutive ways of relating semiotically to the environment 446
and thus accede to more complex relational activities. However, even though pheromones and/or odors 447 might have diminished their primary role as communication tools during evolution, they may still play some 448 important roles in cognitive functions in mammals. Upon attaining higher levels of complexity, due to the 449 development of elaborated sensory apparatuses, these organisms might have gradually acquired the capacity 450 to form fine-tuned internal impressions of their surroundings, with the consequent advantage of entering into 451 more sophisticated Umwelts (Hoffmeyer, 1996). 452
The evolution of complex organisms brought with it the establishment of more complex relations which 453 enabled the interacting parts or partners to explore new dimensions. By doing so, living organisms were 454 given the capacity to discover new functions in order to adjust to new environmental requirements. Where 455 does this bring us, if olfactory perception is considered in this evolutionary perspective? With the advent of 456 higher cognitive faculties, a new dimension was discovered by living organisms. This is the dimension of 457 time, and time has given organisms the ability to explore the possibility of interacting synchronically or 458 diachronically with each other and with the environment. Any new variation in the environment challenges 459 the organism’s capacity to respond with an adequate phenotypic plasticity and to adapt to new and 460 unforeseeable conditions with newly acquired capabilities (Fordyce, 2006). 461
The fact that higher organisms relate to the environment through the perception of a comprehensive 462 smell-scape implies that they are capable of confronting it with a déjà-smell, as Gilbert (2008) calls it, that is 463 to say, with smells already experienced. For this to happen, the olfactory system must be versatile enough to 464 entertain new odor exposures and relate them with past smell experiences. Olfactory receptors may thus be 465 adaptable to new and unpredictable chemical combinations and new odorant molecules perceived as if fuzzy-466 encoded rather than set in a precise key-and-lock mode (Hoare et al. 2008). 467
If these requirements are somehow satisfied, then the opportunity emerges for higher organisms to engage 468 in novel associations between smells and memories. Regardless of whether they are positive or negative, 469 they are nevertheless experiences that may impinge on new social and cultural habits and, with time, be fixed 470 in the species’ behavior (Herz, 2007). It is important to underline at this juncture that the higher one goes in 471 the systemic hierarchy, the more plastic the relation between olfactory perception and the environmental 472 semio-chemical load may become and, consequently, more likely for certain recovered smell memories to be 473 emotionally driven. This makes it possible for certain smell experiences to recall autobiographical memories 474 more effectively than other sensory perceptions (Chu and Downes, 2000). Perhaps the reason for this 475 privileged proto-cognitive faculty is to be found in the ancestry of olfaction as respect to other senses. The 476 olfactory epithelium is the neural network closest to the limbic system and olfactory neurons project to the 477 central nervous system only with unmyelinated axons (Herz and Hengen, 1996). This deeply grounded link 478 between olfaction and proto-cognition is likely to be instantiated in utero where the child sense of smell may 479 constitute the primary communication mechanism connecting him to the mother (Van Toller and Kendal-480 Reed, 1995). 481
In conclusion, taken together, these few examples highlight the importance of relationships with respect 482 to the importance given to the properties of the participating partners. There are certainly mechanisms 483 sustaining energetically a number of biological processes that help us understand how things work; and there 484 are also functions that need to be realized by the bearing organism if it is to successfully face every 485 unpredictable environmental change; but there are also signs that, if properly recognized by an interpretant, 486 make relationships significant on the basis of past experience. None of these explanatory strategies are 487 sufficient in themselves to provide satisfactory structural, functional and behavioral accounts of any living 488 organism. In our view, these strategies may well act to support one another and only when taken together can 489 they provide us with a realistic account of the complexity of life. 490
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