The Role of the Stimulus in Olfactory Plasticity.

Plasticity, the term we use to describe the ability of a nervous system to change with experience, is the evolutionary adaptation that freed animal behavior from the confines of genetic determinism. This capacity, which increases with brain complexity, is nowhere more evident than in vertebrates, especially mammals. Though the scientific study of brain plasticity dates back at least to the mid-19th century, the last several decades have seen unprecedented advances in the field afforded by new technologies. Olfaction is one system that has garnered particular attention in this realm because it is the only sensory modality with a lifelong supply of new neurons, from two niches no less! Here, we review some of the classical and contemporary literature dealing with the role of the stimulus or lack thereof in olfactory plasticity. We have restricted our comments to studies in mammals that have used dual tools of the field: stimulus deprivation and stimulus enrichment. The former manipulation has been implemented most frequently by unilateral naris occlusion and, thus, we have limited our comments to research using this technique. The work reviewed on deprivation provides substantial evidence of activity-dependent processes in both developing and adult mammals at multiple levels of the system from olfactory sensory neurons through to olfactory cortical areas. However, more recent evidence on the effects of deprivation also establishes several compensatory processes with mechanisms at every level of the system, whose function seems to be the restoration of information flow in the face of an impoverished signal. The results of sensory enrichment are more tentative, not least because of the actual manipulation: What odor or odors? At what concentrations? On what schedule? All of these have frequently not been sufficiently rationalized or characterized. Perhaps it is not surprising, then, that discrepant results are common in sensory enrichment studies. Despite this problem, evidence has accumulated that even passively encountered odors can "teach" olfactory cortical areas to better detect, discriminate, and more efficiently encode them for future encounters. We discuss these and other less-established roles for the stimulus in olfactory plasticity, culminating in our recommended "aspirations" for the field going forward.

The sorption/chromatography hypothesis of olfactory discrimination: The rise, fall, and rebirth of a Phoenix.

Herein, I discuss the enduring mystery of the receptor layout in the vertebrate olfactory system. Since the awarding of the 2004 Nobel Prize to Axel and Buck for their discovery of the gene family that encodes olfactory receptors, our field has enjoyed a golden era. Despite this Renaissance, an answer to one of the most fundamental questions for any sensory system-what is the anatomical logic of its receptor array?-eludes us, still, for olfaction! Indeed, the only widely debated hypothesis, finding its origins in the musing of another Nobel laureate Sir Edgar Adrian, has it that the vertebrate nose organizes its receptors according to the "sorptive" properties of their ligands. This idea, known as the "sorption" or "chromatography" hypothesis, enjoys considerable support despite being controversial. Here, I review the history of the hypothesis-its rises and falls-and discuss the latest data and future prospects for this perennial idea whose history I liken to the mythical Phoenix.

The olfactory bulbectomy disease model: A Re-evaluation.

The olfactory bulbectomized rodent has long been one of the preferred animal models of depression and certain other neuropsychiatric diseases. In fact, it is considered unparalleled, by some, in the search for antidepressant medication and the literature generated about the model is prodigious. We have revisited the "syndrome" of behavioral sequela following bulbectomy choosing ecologically valid tests likely to be underpinned with evolutionarily preserved neural circuits. Our test battery included measurements of activity, intermale aggression, pleasure seeking, stress/fear and non-spatial memory. The emphasis was on the timetable of syndrome emergence, since this has been understudied and bears on the widely held belief that non-olfactory effects dominate. Our results largely agree with previous reports describing the behavioral syndrome in that we document bulbectomized mice as hyperactive, non-aggressive and fearless. However, we did not find deficits in memory as have frequently been reported in previous studies. Notably, our results revealed that some syndrome behaviors-including the hallmark of hyperactivity-appear immediately or soon after surgery. This rapid appearance casts doubt on the widely held view that compensatory reorganization of limbic and prefrontal cortical areas following bulbectomy underlies the syndrome. Rather, hyperactivity, non-aggressiveness, reduced fear and diminished sucrose preference in the olfactory bulbectomized mouse find ready explanations in the loss of smell that is the immediate and irreversible outcome of bulbectomy. Finally, after a critical consideration of the literature and our results, we conclude that the olfactory bulbectomy model lacks the validity and simplicity previously credited to it. Indeed, we deem this lesion unsuitable as a model of most neuropsychiatric diseases since its effects are at least as complex and misunderstood as the disorders it is purported to model.

Olfactory Deprivation and Enrichment: An Identity of Opposites?

The effects of deprivation and enrichment on the electroolfactogram of mice were studied through the paradigms of unilateral naris occlusion and odor induction, respectively. Deprivation was shown to cause an increase in electroolfactogram amplitudes after 7 days. We also show that unilateral naris occlusion is not detrimental to the gross anatomical appearance or electroolfactogram of either the ipsilateral or contralateral olfactory epithelium even after year-long survival periods, consistent with our previous assumptions. Turning to induction, the increase in olfactory responses after a period of odor enrichment, could not be shown in CD-1 outbred mice for any odorant tried. However, consistent with classical studies, it was evident in C57BL/6J inbred mice, which are initially insensitive to isovaleric acid. As is the case for deprivation, enriching C57BL/6J mice with isovaleric acid causes an increase in their electroolfactogram response to this odorant over time. In several experiments on C57BL/6J mice, the odorant specificity, onset timing, recovery timing, and magnitude of the induction effect were studied. Considered together, the current findings and previous work from the laboratory support the counterintuitive conclusion that both compensatory plasticity in response to deprivation and induction in response to odor enrichment are caused by the same underlying homeostatic mechanism, the purpose of which is to preserve sensory information flow no matter the odorant milieu. This hypothesis, the detailed evidence supporting it, and speculations concerning human odor induction are discussed.

The effect of odor enrichment on olfactory acuity: Olfactometric testing in mice using two mirror-molecular pairs.

Intelligent systems in nature like the mammalian nervous system benefit from adaptable inputs that can tailor response profiles to their environment that varies in time and space. Study of such plasticity, in all its manifestations, forms a pillar of classical and modern neuroscience. This study is concerned with a novel form of plasticity in the olfactory system referred to as induction. In this process, subjects unable to smell a particular odor, or unable to differentiate similar odors, gain these abilities through mere exposure to the odor(s) over time without the need for attention or feedback (reward or punishment). However, few studies of induction have rigorously documented changes in olfactory threshold for the odor(s) used for "enrichment." We trained 36 CD-1 mice in an operant-olfactometer (go/no go task) to discriminate a mixture of stereoisomers from a lone stereoisomer using two enantiomeric pairs: limonene and carvone. We also measured each subject's ability to detect one of the stereoisomers of each odor. In order to assess the effect of odor enrichment on enantiomer discrimination and detection, mice were exposed to both stereoisomers of limonene or carvone for 2 to 12 weeks. Enrichment was effected by adulterating a subject's food (passive enrichment) with one pair of enantiomers or by exposing a subject to the enantiomers in daily operant discrimination testing (active enrichment). We found that neither form of enrichment altered discrimination nor detection. And this result pertained using either within-subject or between-subject experimental designs. Unexpectedly, our threshold measurements were among the lowest ever recorded for any species, which we attributed to the relatively greater amount of practice (task replication) we allowed our mice compared to other reports. Interestingly, discrimination thresholds were no greater (limonene) or only modestly greater (carvone) from detection thresholds suggesting chiral-specific olfactory receptors determine thresholds for these compounds. The super-sensitivity of mice, shown in this study, to the limonene and carvone enantiomers, compared to the much lesser acuity of humans for these compounds, reported elsewhere, may resolve the mystery of why the former group with four-fold more olfactory receptors have tended, in previous studies, to have similar thresholds to the latter group. Finally, our results are consistent with the conclusion that supervised-perceptual learning i.e. that involving repeated feedback for correct and incorrect decisions, rather than induction, is the form of plasticity that allows animals to fully realize the capabilities of their olfactory system.

Tests of the chromatographic theory of olfaction with highly soluble odors: a combined electro-olfactogram and computational fluid dynamics study in the mouse.

The idea that the vertebrate nasal cavity operates like a gas chromatograph to separate and discriminate odors, referred to herein as the 'chromatographic theory' (CT), has a long and interesting history. Though the last decade has seen renewed interest in the notion, its validity remains in question. Here we examine a necessary condition of the theory: a correlation between nasal odor deposition patterns based on mucus solubility and the distribution of olfactory sensory neuron odotypes. Our recent work in the mouse failed to find such a relationship even across large sorption gradients within the olfactory epithelium (OE). However, these studies did not test extremely soluble odorants or low odor concentrations, factors that could explain our inability to find supporting evidence for the CT. The current study combined computational fluid dynamics (CFD) simulations of odor sorption patterns and electro-olfactogram (EOG) measurements of olfactory sensory neuron responses. The odorants tested were at the extremes of mucus solubility and at a range of concentrations. Results showed no relationship between local odor sorption patterns and EOG response maps. Together, results again failed to support a necessary condition of the CT casting further doubt on the viability of this classical odor coding mechanism.

Forever young: Neoteny, neurogenesis and a critique of critical periods in olfaction.

The critical period concept has been one of the most transcendent in science, education, and society forming the basis of our fixation on 'quality' of childhood experiences. The neural basis of this process has been revealed in developmental studies of visual, auditory and somatosensory maps and their enduring modification through manipulations of experience early in life. Olfaction, too, possesses a number of phenomena that share key characteristics with classical critical periods like sensitive temporal windows and experience dependence. In this review, we analyze the candidate critical period-like phenomena in olfaction and find them disanalogous to classical critical periods in other sensory systems in several important ways. This leads us to speculate as to why olfaction may be alone among exteroceptive systems in lacking classical critical periods and how life-long neurogenesis of olfactory sensory neurons and bulbar interneurons-a neotenic vestige-- relates to the structure and function of the mammalian olfactory system.

Odor-Cued Bitter Taste Avoidance.

In the course of our ongoing studies of odor-cued taste avoidance (OCTA) to measure olfactory capabilities in animals, we observed that mice could rapidly learn to use the vapor of the classical bitterant quinine hydrochloride to avoid contact with the tastant. Here we expand on this observation to determine which among several compounds generally classed as bitter could be detected at a distance. Since mice were initially naïve we were able to assess whether the vapors of the bitter compounds tested were innately aversive as are their tastes. CD-1 mice could readily use vapor cues from quinine hydrochloride, denatonium benzoate (DB), and 6-propyl-2-thiouracil to avoid their taste. Although mice did not hesitate to make contact with these solutions on their first exposure, they did learn to do so typically after only 1 or 2 exposures. Bilaterally bulbectomized mice did not learn or retain the ability to avoid quinine and DB solutions by vapor alone, implicating olfaction as the mode of detection. Saturated aqueous solutions of sucrose octaacetate and caffeine which are bitter to humans and some strains of mice were not aversive in our studies. The very low vapor concentrations of the 3 bitterant solutions that mice detected at a distance, suggest that impurities in the reagent grade solutions, rather than the bitter molecules themselves were the basis of detection. Implications of these findings for taste testing and the role of odor in food acceptance/rejections decisions are discussed.

Tests of the sorption and olfactory "fovea" hypotheses in the mouse.

The spatial distribution of receptors within sensory epithelia (e.g., retina and skin) is often markedly nonuniform to gain efficiency in information capture and neural processing. By contrast, odors, unlike visual and tactile stimuli, have no obvious spatial dimension. What need then could there be for either nearest-neighbor relationships or nonuniform distributions of receptor cells in the olfactory epithelium (OE)? Adrian (Adrian ED. J Physiol 100: 459-473, 1942; Adrian ED. Br Med Bull 6: 330-332, 1950) provided the only widely debated answer to this question when he posited that the physical properties of odors, such as volatility and water solubility, determine a spatial pattern of stimulation across the OE that could aid odor discrimination. Unfortunately, despite its longevity, few critical tests of the "sorption hypothesis" exist. Here we test the predictions of this hypothesis by mapping mouse OE responses using the electroolfactogram (EOG) and comparing these response "maps" to computational fluid dynamics (CFD) simulations of airflow and odorant sorption patterns in the nasal cavity. CFD simulations were performed for airflow rates corresponding to quiet breathing and sniffing. Consistent with predictions of the sorption hypothesis, water-soluble odorants tended to evoke larger EOG responses in the central portion of the OE than the peripheral portion. However, sorption simulation patterns along individual nasal turbinates for particular odorants did not correlate with their EOG response gradients. Indeed, the most consistent finding was a rostral-greater to caudal-lesser response gradient for all the odorants tested that is unexplained by sorption patterns. The viability of the sorption and related olfactory "fovea" hypotheses are discussed in light of these findings.NEW & NOTEWORTHY Two classical ideas concerning olfaction's receptor-surface two-dimensional organization-the sorption and olfactory fovea hypotheses-were found wanting in this study that afforded unprecedented comparisons between electrophysiological recordings in the mouse olfactory epithelium and computational fluid dynamic simulations of nasal airflow. Alternatively, it is proposed that the olfactory receptor layouts in macrosmatic mammals may be an evolutionary contingent state devoid of the functional significance found in other sensory epithelia like the cochlea and retina.

Compensatory plasticity in the olfactory epithelium: age, timing, and reversibility.

Like other biological systems, olfaction responds "homeostatically" to enduring change in the stimulus environment. This adaptive mechanism, referred to as compensatory plasticity, has been studied almost exclusively in developing animals. Thus it is unknown if this phenomenon is limited to ontogenesis and irreversible, characteristics common to some other forms of plasticity. Here we explore the effects of odor deprivation on the adult mouse olfactory epithelium (OE) using nasal plugs to eliminate nasal airflow unilaterally. Plugs were in place for 2-6 wk after which electroolfactograms (EOGs) were recorded from the occluded and open sides of the nasal cavity. Mean EOG amplitudes were significantly greater on the occluded than on the open side. The duration of plugging did not affect the results, suggesting that maximal compensation occurs within 2 wk or less. The magnitude of the EOG difference between the open and occluded side in plugged mice was comparable to adults that had undergone surgical naris occlusion as neonates. When plugs were removed after 4 wk followed by 2 wk of recovery, mean EOG amplitudes were not significantly different between the always-open and previously plugged sides of the nasal cavity suggesting that this form of plasticity is reversible. Taken together, these results suggest that compensatory plasticity is a constitutive mechanism of olfactory receptor neurons that allows these cells to recalibrate their stimulus-response relationship to fit the statistics of their current odor environment.

Odor-cued taste avoidance: a simple and robust test of mouse olfaction.

In odor-cued taste avoidance (OCTA), thirsty mice, offered either an odorized nonaversive fluid (S+) or an odorized aversive fluid (S-), quickly learn to use odor to avoid drinking the S-. Acquisition of both odor detection and odor discrimination tasks is very rapid with learning evidenced in most cases by either long response times or total avoidance on the second presentation of the S- stimulus. OCTA is perhaps one of the simplest conditioning procedures for assessing olfaction in mice; it requires only a test box, drinkometer circuit, and thirsty mice accustomed to drinking in the apparatus. Its advantages over the most commonly used alternatives, habituation-dishabituation, and the mouse dig test, are discussed.

The effects of naris occlusion on mouse nasal turbinate development.

Unilateral naris occlusion, a standard method for causing odor deprivation, also alters airflow on both sides of the nasal cavity. We reasoned that manipulating airflow by occlusion could affect nasal turbinate development given the ubiquitous role of environmental stimuli in ontogenesis. To test this hypothesis, newborn mice received unilateral occlusion or sham surgery and were allowed to reach adulthood. Morphological measurements were then made of paraffin sections of the whole nasal cavity. Occlusion significantly affected the size, shape and position of turbinates. In particular, the nasoturbinate, the focus of our quantitative analysis, had a more delicate appearance on the occluded side relative to the open side. Occlusion also caused an increase in the width of the dorsal meatus within the non-occluded and occluded nasal fossae, compared with controls, and the position of most turbinates was altered. These results suggest that a mechanical stimulus from respiratory airflow is necessary for the normal morphological development of turbinates. To explore this idea, we estimated the mechanical forces on turbinates caused by airflow during normal respiration that would be absent as a result of occlusion. Magnetic resonance imaging scans were used to construct a three-dimensional model of the mouse nasal cavity that provided the input for a computational fluid dynamics simulation of nasal airflow. The simulation revealed maximum shear stress values for the walls of turbinates in the 1 Pa range, a magnitude that causes remodeling in other biological tissues. These observations raise the intriguing possibility that nasal turbinates develop partly under the control of respiratory mechanical forces.

Studies of olfactory system neural plasticity: the contribution of the unilateral naris occlusion technique.

Unilateral naris occlusion has long been the method of choice for effecting stimulus deprivation in studies of olfactory plasticity. A significant body of literature speaks to the myriad consequences of this manipulation on the ipsilateral olfactory pathway. Early experiments emphasized naris occlusion's deleterious and age-critical effects. More recent studies have focused on life-long vulnerability, particularly on neurogenesis, and compensatory responses to deprivation. Despite the abundance of empirical data, a theoretical framework in which to understand the many sequelae of naris occlusion on olfaction has been elusive. This paper focuses on recent data, new theories, and underappreciated caveats related to the use of this technique in studies of olfactory plasticity.

The effects of unilateral naris occlusion on gene expression profiles in mouse olfactory mucosa.

Unilateral naris occlusion has been the method of choice for effecting stimulus deprivation in studies of olfactory plasticity. Early experiments emphasized the deleterious effects of this technique on the developing olfactory system while more recent studies have pointed to several apparently "compensatory" responses. However, the evidence for deprivation-induced compensatory processes in olfaction remains fragmentary. High-throughput methods such as microarray analysis can help fill the deficits in our understanding of naris occlusion as a mode of stimulus deprivation. Here we report for young adult mice the effects of early postnatal naris occlusion on the olfactory mucosal transcriptome using microarray analysis with RT-PCR confirmation. The transcripts of key genes involved in olfactory reception, transduction, and transmission were up-regulated in deprived-side olfactory mucosa, with opposite effects in non-deprived-side mucosa, compared to controls. Results support the hypothesis that odor environment triggers a previously unknown homeostatic control mechanism in olfactory receptor neurons designed to maximize information transfer.

Universality in the evolution of orientation columns in the visual cortex.

The brain's visual cortex processes information concerning form, pattern, and motion within functional maps that reflect the layout of neuronal circuits. We analyzed functional maps of orientation preference in the ferret, tree shrew, and galago--three species separated since the basal radiation of placental mammals more than 65 million years ago--and found a common organizing principle. A symmetry-based class of models for the self-organization of cortical networks predicts all essential features of the layout of these neuronal circuits, but only if suppressive long-range interactions dominate development. We show mathematically that orientation-selective long-range connectivity can mediate the required interactions. Our results suggest that self-organization has canalized the evolution of the neuronal circuitry underlying orientation preference maps into a single common design.

How does long-term odor deprivation affect the olfactory capacity of adult mice?

BACKGROUND: Unilateral naris occlusion (UNO) has been the most common method of effecting stimulus deprivation in studies of olfactory plasticity. However, despite the large corpus on the effects of this manipulation, dating back to the 19th century, little is known about its behavioral sequela. Here we report the results of standard olfactory habituation and discrimination studies on adult mice that had undergone perinatal UNO followed by adult contralateral olfactory bulbectomy (bulb-x). METHODS: The olfactory performance of UNO mice was compared to matched controls that had unilateral bulb-x but open nares. Both habituation and discrimination (operant) experiments employed a protocol in which after successful dishabituation or discrimination to dilute individual odors (A = 0.01% isoamyl acetate; B = 0.01% ethyl butyrate; each v/v in mineral oil), mice were challenged with a single odor versus a mixture comparison (A vs. A + B). In a series of tests the volume portion of Odor B in the mixture was systematically decreased until dishabituation or discrimination thresholds were reached. RESULTS: For the habituation experiment, UNOs (n = 10) and controls (n = 9) dishabituated to a 10% mixture of Odor B in Odor A after being habituated to A alone, while both groups failed to show differential responding to a 2% mixture of B in A. However, the UNO group's increased investigation durations for the 2% mixture approached significance (p < 0.06). A replication of this study (7 controls & 8 UNOs) confirmed that controls did not differentiate Odor A and a 2% mixture of B in A but UNOs did not (p < 0.05). For the discrimination experiment, 4 UNOs and 4 controls were shaped to dig in one of two containers of sand that contained the S+ odor (Odor B) to obtain sugar pellet rewards. As in the habituation experiment, UNOs displayed greater olfactory capacity than controls on this task. Controls and UNOs had an average mixture discrimination threshold of 1.6% (+/- 0.4) and 0.22% (+/- 0.102) respectively, a difference that was statistically significant (p < 0.02). CONCLUSIONS: Adult mice relying on an olfactory system deprived of odor by naris occlusion from near the time of birth display enhanced olfactory capacity compared to control mice. This counterintuitive result suggests that UNO is neither an absolute method of deprivation nor does it diminish olfactory capabilities. Enhanced olfactory capacity, as observed in the current study, that is a consequence of deprivation, is consistent with recent molecular and physiological evidence that stimulus deprivation triggers compensatory processes throughout the olfactory system.

Naris occlusion alters the electro-olfactogram: evidence for compensatory plasticity in the olfactory system.

Unilateral naris occlusion (NO) has been widely used as a method of olfactory deprivation to study the role of stimulus-driven activity in olfactory development [P.C. Brunjes, Unilateral naris closure and olfactory system development, Brain Res. Rev. 19 (1994) 146-160]. Recent immunochemical studies of the olfactory epithelium (OE) following NO provide evidence for a previously unknown compensatory response to deprivation [D.M. Coppola, A. Waguespack, M. Reems, M.L. Butman, J. Cherry, Naris occlusion alters transductory protein immunoreactivity in olfactory epithelium, Histol. Histopathol. 21 (2006) 487-501; A. Waguespack, M. Reems, M.L. Butman, J. Cherry, D.M. Coppola, Olfactory receptor neurons have enhanced olfactory marker protein immunoreactivity in the nasal cavity ipsilateral to naris occlusion, Brain Res. 1044 (2005) 1-7]. To further investigate this phenomenon we measured electro-olfactograms (EOG) from the open and occluded OE of adult mice that had undergone NO as newborns. EOG waveforms from the open side OE of NO mice were indistinguishable from those obtained from untreated animals. However, amplitudes of EOGs from the occluded OE of NO mice were greater, on average, than those recorded at matched locations from the open side. This result was consistent across turbinates, odors, and all but the highest odor concentration. In addition, EOGs recorded from the occluded OE had significantly slower onset and recovery kinetics. Responses in a double-pulse protocol confirmed that the kinetics of the cellular or population processes that underlie the EOG are slowed by NO. These results provide the most direct support, to date, for compensatory plasticity in the olfactory system.

Naris occlusion alters olfactory marker protein immunoreactivity in olfactory epithelium.

Though its function remains obscure, olfactory marker protein (OMP) has been implicated in olfactory transduction and the enhancement of neurogenesis within olfactory epithelium. Here we show, using Western blot analysis and immunocytochemistry, that unilateral naris occlusion (UNO) on postnatal day 1 alters OMP immunoreactivity (IR) differentially on the occluded and non-occluded sides of the nasal cavity in 18, 24 and 70-day-old mice. Compared to untreated animals, UNO-treated animals had a decrease in OMP-IR in olfactory receptor neurons on the non-occluded side and an increase in OMP-IR in olfactory receptor neurons on the occluded side of the nasal cavity. These results suggest that OMP concentration is up- or down-regulated depending on the amount of odor stimulation olfactory receptor neurons receive. It is proposed that this apparent change in protein concentration may be part of a more general compensatory response by olfactory neurons to levels of odor in the environment.

Visual experience promotes the isotropic representation of orientation preference.

Within the visual cortex of several mammalian species, more circuitry is devoted to the representation of vertical and horizontal orientations than oblique orientations. The sensitivity of this representation of orientation preference to visual experience during cortical maturation and the overabundance of cardinal contours in the environment suggest that vision promotes the development of this cortical anisotropy. We tested this idea by measuring the distribution of cortical orientation preference and the degree of orientation selectivity in developing normal and dark-reared ferrets using intrinsic signal optical imaging. The area of the angle map of orientation preference representing cardinal and oblique orientations was determined; in addition, orientation selectivity indices were computed separately for cardinal and oblique difference images. In normal juvenile animals, we confirm a small, but statistically significant overrepresentation of near horizontal orientations in the cortical angle map. However, the degree of anisotropy did not increase in the weeks that followed eye opening when orientation selectivity matured; rather, it decreased. In dark-reared ferrets, an even greater cortical anisotropy emerged, but angle maps in these animals developed an apparently anomalous overrepresentation of near vertical orientations. Thus, the overrepresentation of cardinal orientations in the visual cortex does not require experience with an anisotropic visual environment; indeed, cortical anisotropy can develop in the complete absence of vision. These observations suggest that the role of visual experience in cortical maturation is to promote the isotropic representation of orientation preference.