Trial-by-trial discrimination of three enantiomer pairs by neural ensembles in mammalian olfactory bulb

Populations of output neurons in the mammalian olfactory bulb (OB) exhibit distinct, widespread spatial and temporal activation patterns when stimulated with odorants. However, questions remain as to how ensembles of mitral/tufted (M/T) neurons in the mammalian OB represent odorant information. In this report, the single-trial encoding limits of random ensembles of putative single- and multiunit M/T cells in the anesthetized rat OB during presentations of enantiomers of limonene, carvone, and 2-butanol are investigated using simultaneous multielectrode recording techniques. The results of these experiments are: the individual constituents of our recorded ensembles broadly represent information about the presented odorants, the ensemble single-trial response of small spatially distributed populations of M/T neurons can readily discriminate between six different odorants, and the most consistent odorant discrimination is attained when the ensemble consists of all available units and their responses are integrated over an entire breathing cycle. These results suggest that small differences in spike counts among the ensemble members become significant when taken within the context of the entire ensemble. This may explain how ensembles of broadly tuned OB neurons contribute to olfactory perception and may explain how small numbers of individual units receiving input from distinct olfactory receptor neurons can be combined to form a robust representation of odorants.     

Molecular features of odorants systematically influence slow temporal responses across clusters of coordinated antennal lobe units in the moth Manduca sexta

Behavioral studies of olfactory discrimination and stimulus generalization in many species indicate that the molecular features of monomolecular odorants are important for odor discrimination. Here we evaluate how features, such as carbon chain length and functional group, are represented in the first level of synaptic processing. We recorded antennal lobe ensemble responses in the moth Manduca sexta to repeated 100-ms pulses of monomolecular alcohols and ketones. Most units exhibited a significant change in spike rate in response to most odorants that outlasted the duration of the stimulus. Peristimulus data were then sampled over 780 ms for each pulse of all odorants. Factor analysis was used to assess whether there were groups of units with common response patterns. We found that factors identified and represented activity for clusters of units with common temporal response characteristics. These temporally patterned responses typically spanned 780 ms and were often dependent on carbon chain length and functional group. Furthermore, cross-correlation analysis frequently indicated significant coincident spiking even during spontaneous activity. However, this synchrony occurred mainly between units recorded on the same tetrode. In a final analysis, the Euclidean distance between odor responses was calculated for each pair of odorants using factors as dimensions. The distance between responses for any two odorants was maximized by approximately 240 ms. This time course corresponded to the brief sequence of coordinated bursts across the recorded population. The distance during this period was also a function of systematic differences in molecular features. Results of this Euclidian analysis thus directly correlate to previous behavioral studies of stimulus generalization in M. sexta.     

Age-associated loss of selectivity in human olfactory sensory neurons

We report a cross-sectional study of olfactory impairment with age based on both odorant-stimulated responses of human olfactory sensory neurons (OSNs) and tests of olfactory threshold sensitivity. A total of 621 OSNs from 440 subjects in 2 age groups of younger (≤ 45 years) and older (≥ 60 years) subjects were investigated using fluorescence intensity ratio fura-2 imaging. OSNs were tested for responses to 2 odorant mixtures, as well as to subsets of and individual odors in those mixtures. Whereas cells from younger donors were highly selective in the odorants to which they responded, cells from older donors were more likely to respond to multiple odor stimuli, despite a loss in these subjects' absolute olfactory sensitivity, suggesting a loss of specificity. This degradation in peripheral cellular specificity may impact odor discrimination and olfactory adaptation in the elderly. It is also possible that chronic adaptation as a result of reduced specificity contributes to observed declines in absolute sensitivity.     

Modulation of odor-induced increases in [Ca(2+)](i) by inhibitors of protein kinases A and C in rat and human olfactory receptor neurons

Protein kinases A and C have been postulated to exert multiple effects on different elements of signal transduction pathways in olfactory receptor neurons. However, little is known about the modulation of olfactory responses by protein kinases in intact olfactory receptor neurons. To further elucidate the details of the modulation of odorant responsiveness by these protein kinases, we investigated the action of two protein kinase inhibitors: H89, an inhibitor of protein kinase A, and N-myristoylated EGF receptor, an inhibitor of protein kinase C, on odorant responsiveness in intact olfactory neurons. We isolated individual olfactory neurons from the adult human and rat olfactory epithelium and measured responses of the isolated cells to odorants or biochemical activators that have been shown to initiate cyclic AMP or inositol 1,4,5-trisphospate production in biochemical preparations. We employed calcium imaging techniques to measure odor-elicited changes in intracellular calcium that occur over several seconds. In human olfactory receptor neurons, the protein kinase A and C inhibitors affected the responses to different sets of odorants. In rats, however, the protein kinase C inhibitor affected responses to all odorants, while the protein kinase A inhibitor had no effect. In both species, the effect of inhibition of protein kinases was to enhance the elevation and block termination of intracellular calcium levels elicited by odorants.Our results show that protein kinases A and C may modulate odorant responses of olfactory neurons by regulating calcium fluxes that occur several seconds after odorant stimulation. The effects of protein kinase C inhibition are different in rat and human olfactory neurons, indicating that species differences are an important consideration when applying data from animal studies to apply to humans.     

Odor representation and discrimination in mitral/tufted cells of the rat olfactory bulb

Extracellular single-unit responses to odorants with various properties were recorded from mitral/tufted cells over large areas of the olfactory bulb of anesthetized rats. Each cell was exposed to one stimulus set consisting of five different odorants each at five concentrations. The resulting concentration-response profiles were compared. All mitral/tufted cells examined responded to two or more odorants, and the largest proportion of the cells were sensitive to all five odorants. Cells unresponsive to all five odorants regardless of concentration were not observed. Mitral/tufted cells sensitive to all three of the odorants that are known to evoke maximal electro-olfactograms in different regions of the olfactory epithelium were distributed widely throughout the olfactory bulb. There were no significant differences in latencies of odor responses either across recording sites or across odorants. A comparison of the concentration-response profiles suggested that all of the mitral/tufted cells were equally capable of responding to any odorant with their own distinctive pattern, but that the cells tended to show an identical pattern rather than variable pattern of response to different odorants. Five mitral/tufted cells isolated within 800 m of one electrode track showed different concentration-response profiles. Of 18 simultaneously recorded spike pairs with different amplitudes and discharge patterns recorded incidentally through one electrode at different sites, 10 had different and 8 had identical response patterns to odorants. These results suggest that: (1) mitral/tufted cells are sensitive to a broad spectrum of odorants, but respond with their own patterns to odorants; (2) odor discrimination is not uniform in neighboring cells, and a discrimination unit is comprised of a single cell.     

Gonadotropin releasing hormone (GnRH) modulates odorant responses in the peripheral olfactory system of axolotls

Peripheral signal modulation plays an important role in sensory processing. Activity in the vertebrate olfactory epithelium may be modulated by peptides released from the terminal nerve, such as gonadotropin releasing hormone (GnRH). Here, we demonstrate that GnRH modulates odorant responses in aquatic salamanders (axolotls, Ambystoma mexicanum). We recorded electrical field potentials (electro-olfactograms, or EOGs) in response to stimulation with four different amino acid odorants, L-lysine, L-methionine, L-cysteine, and L-glutamic acid. EOG responses were recorded from the main olfactory epithelium before, during, and after application of 10 microM GnRH. This protocol was repeated for a total of three trials with 60-80 min between trials. The effect of GnRH on EOG responses was broadly similar across odorants and across trials. In general, EOG responses were reduced to 79% of the initial magnitude during application of GnRH; in some trials in which glutamic acid served as the odorant, EOG responses were enhanced during the wash period. Although the 4-min inter-stimulus interval did not lead to adaptation of EOG responses during the first trial, we frequently observed evidence of adaptation during the second and third trials. In addition, we found that lower concentrations of GnRH produced a smaller effect. These results demonstrate that GnRH can modulate odorant responses in the peripheral olfactory system.     

Discrimination among odorants by single neurons of the rat olfactory bulb

1. Intracellular and extracellular recordings were made from rat olfactory bulb mitral and tufted cells during odor stimulation and during electrical stimulation of the olfactory nerve. Neurons were identified by horseradish peroxidase injections and/or antidromic activation. The presentation of multiple concentrations of at least one odorant in a cyclic artificial sniff paradigm, as reported previously (10), allowed the study of odor responses. This approach was extended to multiple odorants to compare their concentration-response profiles. This procedure avoids the problems of interpretation resulting from nonequivalence of the effective concentrations of different odorants used as stimuli that have characterized previous studies of odor quality effects. Comparisons of intracellular events and responses to electrical stimulation with the odor-induced spike train activity allow us to begin to delineate the local circuitry involved in generating odor-induced responses. 2. The concentration-response profiles of the 72 cells in the present study are comparable to those previously reported for output neurons of the olfactory bulb, showing ordered changes in the temporal patterning of spike activity with step changes in odor concentration. However, eight of the neurons exhibited inhibitory responses to lower concentrations, but excitation, at similar latency, to higher concentrations of the same odorant. These data emphasize that to study pattern changes induced by changing odor quality the influence of stimulus intensity must also be carefully examined. The data also provide evidence that the temporal pattern evoked by an odorant is probably not in itself the code for odor quality recognition. 3. Complete concentration-response profiles, including subthreshold concentrations, to more than one odorant show that, although responses to the different odorant can evolve systematically with concentration, the responses to different odorants can evolve through very different patterns. For example, in some cells, the response patterns to different odors were complementary in form. These results demonstrate that the patterned responses of olfactory bulb neurons can reflect changes in odor quality as well as intensity. 4. Intracellular recording was employed to compare the temporal patterning of spikes during odor stimulation with membrane potential changes. In some cases, the spike pattern was closely correlated with apparent postsynaptic potentials. However, there were several clear exceptions. In five cells, a prominent hyperpolarization, seen in the first sniff of a series of 10 consecutive sniffs, was associated with pauses in spike activity. In the following     

Maps of odorant molecular features in the Mammalian olfactory bulb

The olfactory bulb (OB) is the first relay station of the central olfactory system in the mammalian brain and contains a few thousand glomeruli on its surface. Because individual glomeruli represent a single odorant receptor, the glomerular sheet of the OB forms odorant receptor maps. This review summarizes the emerging view of the spatial organization of the odorant receptor maps. Recent studies suggest that individual odorant receptors are molecular-feature detecting units, and so are individual glomeruli in the OB. How are the molecular-feature detecting units spatially arranged in the glomerular sheet? To characterize the molecular-feature specificity of an individual glomerulus, it is necessary to determine the molecular receptive range (MRR) of the glomerulus and to compare the molecular structure of odorants within the MRR. Studies of the MRR mapping show that 1) individual glomeruli typically respond to a range of odorants that share a specific combination of molecular features, 2) each glomerulus appears to be unique in its MRR property, and 3) glomeruli with similar MRR properties gather together in proximity and form molecular-feature clusters. The molecular-feature clusters are located at stereotypical positions in the OB and might be part of the neural representation of basic odor quality. Detailed studies suggest that the glomerular sheet represents the characteristic molecular features in a systematic, gradual, and multidimensional fashion. The molecular-feature maps provide a basis for understanding how the olfactory cortex reads the odor maps of the OB.     

Chemical factors determine olfactory system beta oscillations in waking rats

Recent studies have pointed to olfactory system beta oscillations of the local field potential (15-30 Hz) and their roles both in learning and as specific responses to predator odors. To describe odorant physical properties, resultant behavioral responses and changes in the central olfactory system that may induce these oscillations without associative learning, we tested rats with 26 monomolecular odorants spanning 6 log units of theoretical vapor pressure (estimate of relative vapor phase concentration) and 10 different odor mixtures. We found odorant vapor phase concentration to be inversely correlated with investigation time on the first presentation, after which investigation times were brief and not different across odorants. Analysis of local field potentials from the olfactory bulb and anterior piriform cortex shows that beta oscillations in waking rats occur specifically in response to the class of volatile organic compounds with vapor pressures of 1-120 mmHg. Beta oscillations develop over the first three to four presentations and are weakly present for some odorants in anesthetized rats. Gamma oscillations show a smaller effect that is not restricted to the same range of odorants. Olfactory bulb theta oscillations were also examined as a measure of effective afferent input strength, and the power of these oscillations did not vary systematically with vapor pressure, suggesting that it is not olfactory bulb drive strength that determines the presence of beta oscillations. Theta band coherence analysis shows that coupling strength between the olfactory bulb and piriform cortex increases linearly with vapor phase concentration, which may facilitate beta oscillations above a threshold.     

Respiratory-phase-related coding of olfactory information in the olfactory bulb of awake freely-breathing rabbits

The "across-neuron pattern" theory of olfactory information coding states that odorants are represented by spatial patterns of activity produced across-neurons in the olfactory bulbs. The present study analyzes the different response patterns encoding olfactory information in both the inspiratory and expiratory phases of the respiratory cycles of awake freely-breathing rabbits. We recorded the single-unit responses of 31 neurons to 5 different odorants, 3 of which were repeatedly delivered 5 times each. These responses were characterized by the change in their mean firing activity during the inspiratory and expiratory phases of the respiratory cycles respectively. Their processing using principal component analyses revealed that both the inspiration- and the expiration-related across-neuron profiles of responses contained information appropriate to the specification of each odorant. It was therefore concluded that there was a dynamic representation of odorants in the olfactory bulb of awake freely-breathing animals. This alternate representation of odorants by two profiles and its physiological significance are discussed.     

A pharmacological profile of the aldehyde receptor repertoire in rat olfactory epithelium

Several lines of evidence suggest that odorants are recognized through a combinatorial process in the olfactory system; a single odorant is recognized by multiple receptors and multiple odorants are recognized by the same receptor. However few details of how this might actually function for any particular odour set or receptor family are available. Approaching the problem from the ligands rather than the receptors, we used the response to a common odorant, octanal, as the basis for defining multiple receptor profiles. Octanal and other aldehydes induce large EOG responses in the rodent olfactory epithelium, suggesting that these compounds activate a large number of odour receptors (ORs). Here, we have determined and compared the pharmacological profile of different octanal receptors using Ca²⁺ imaging in isolated olfactory sensory neurones (OSNs). It is believed that each OSN expresses only one receptor, thus the response profile of each cell corresponds to the pharmacological profile of one particular receptor. We stimulated the cells with a panel of nine odorants, which included octanal, octanoic acid, octanol and cinnamaldehyde among others (all at 30μ m ). Cluster analysis revealed several distinct pharmacological profiles for cells that were all sensitive to octanal. Some receptors had a broad molecular range, while others were activated only by octanal. Comparison of the profiles with that of the one identified octanal receptor, OR-I7, indicated several differences. While OR-I7 is activated by low concentrations of octanal and blocked by citral, other receptors were less sensitive to octanal and not blocked by citral. A lower estimate for the maximal number of octanal receptors is between 33 and 55. This large number of receptors for octanal suggests that, although the peripheral olfactory system is endowed with high sensitivity, discrimination among different compounds probably requires further central processing.     

Neuronal activity of mitral-tufted cells in awake rats during passive and active odorant stimulation

Odorants induce specific modulation of mitral/tufted (MT) cells' firing rate in the mammalian olfactory bulb (OB), inducing temporal patterns of neuronal discharge embedded in an oscillatory local field potential (LFP). While most studies have examined anesthetized animals, little is known about the firing rate and temporal patterns of OB single units and population activity in awake behaving mammals. We examined the firing rate and oscillatory activity of MT cells and LFP signals in behaving rats during two olfactory tasks: passive exposure (PE) and two-alternative (TA) choice discrimination. MT inhibitory responses are predominant in the TA task (76.5%), whereas MT excitatory responses predominate in the PE task (59.2%). Rhythmic discharge in the 12- to 100-Hz range was found in 79.0 and 68.9% of MT cells during PE and TA tasks, respectively. Most odorants presented in PE task increase rhythmic discharges at frequencies >50 Hz, whereas in TA, one of four odorants produced a modest increment <40 Hz. LFP oscillations were clearly modulated by odorants during the TA task, increasing their oscillatory power at frequencies centered at 20 Hz and decreasing power at frequencies >50 Hz. Our results indicate that firing rate responses of MT cells in awake animals are behaviorally modulated with inhibition being a prominent feature of this modulation. The occurrence of oscillatory patterns in single- and multiunitary discharge is also related to stimulation and behavioral context, while the oscillatory patterns of the neuronal population showed a strong dependence on odorant stimulation.     

Odorant exposure increases olfactory sensitivity: olfactory epithelium is implicated

Exposure-induced shifts in sensitivity to odors may involve peripheral and/or central components of the olfactory system. The ability to disconnect the olfactory epithelium from the bulbs provides a unique opportunity to examine how odorant exposure affects each component. In one experiment, odor thresholds were established for either amyl acetate or androstenone. The mice were then exposed for 10 days to the same test odorant for which a threshold was obtained. After exposure, sensitivity to the odorant increased relative to preexposure levels. The mice then underwent bilateral olfactory nerve transection (BNX). When both groups of mice were tested 45-50 days after recovery from surgery and return of olfactory function, increased sensitivity to the exposed odorant persisted; however, 121-203 days after surgery, sensitivity returned to preexposure levels. Another experiment was similar to the first except that mice were exposed to an odorant, either amyl acetate or androstenone, for 10 days beginning 1 day after BNX or sham surgery. When the mice were tested 45-50 days after surgery, sensitivity to the exposed odorant was increased relative to preexposure levels, whereas sensitivity to the nonexposed odorant remained at preexposure levels. Although further work is needed to determine the precise mechanism(s) underlying shifts in sensitivity to odors, these studies provide additional evidence for peripheral involvement in exposure-induced sensitization to odorants and demonstrate the remarkable capacity of the olfactory system to maintain or even regain sensitivity after injury.     

Dense representation of natural odorants in the mouse olfactory bulb

In mammals, odorant molecules are thought to activate only a few glomeruli, leading to the hypothesis that odor representation in the olfactory bulb is sparse. However, the studies supporting this model used anesthetized animals or monomolecular odorants at limited concentration ranges. Using optical imaging and two-photon microscopy, we found that natural odorants at their native concentrations could elicit dense representations in the olfactory bulb. Both anesthesia and odorant concentration were found to modulate the representation density of natural odorants.     

A chemical modification approach to the olfactory code: vapor phase labeling using photoaffinity odorants

A photoaffinity labeling technique was used to study the receptors involved in the discrimination of odorants. Aromatic azides, 1-azidonaphthalene (AzN) and 1-azido-4-nitronaphthalene (AsNN), were found to be pleasant-smelling compounds and produced good responses, giving standard EOG's (electro-olfactogram) of the kind observed for normal odorants. Following irradiation of the frog olfactory mucosa with light during constant stimulation with one of the azides vapor, there was a specific partial inhibition of the receptors for that odorant. The extent of reduction in amplitude of the EOG responses to AzN and AzNN varied between 40 to 60% of the original amplitude.     

Behavioral models of odor similarity

Carbon chain length in several classes of straight-chain aliphatic odorants has been proposed as a model axis of similarity for olfactory research, on the basis of successes of studies in insect and vertebrate species. To assess the influence of task on measured perceptual similarities among odorants and to demonstrate that the systematic similarities observed within homologous odorant series are not task specific, the authors compare 3 different behavioral paradigms for rats (olfactory habituation, generalization, and discrimination). Although overall patterns of odorant similarity are consistent across all 3 of these paradigms, both quantitative measurements of perceptual similarity and comparability with 2-deoxyglucose imaging data from the olfactory bulb are dependent on the specific behavioral tasks used. Thus, behavioral indices of perceptual similarity are affected by task parameters such as learning and reward associations.     

Mitral and tufted cells differ in the decoding manner of odor maps in the rat olfactory bulb

Mitral and tufted cells in the mammalian olfactory bulb are principal neurons, each type having distinct projection pattern of their dendrites and axons. The morphological difference suggests that mitral and tufted cells are functionally distinct and may process different aspects of olfactory information. To examine this possibility, we recorded odorant-evoked spike responses from mitral and middle tufted cells in the aliphatic acid- and aldehyde-responsive cluster at the dorsomedial part of the rat olfactory bulb. Homologous series of aliphatic acids and aldehydes were used for odorant stimulation. In response to adequate odorants, mitral cells showed spike responses with relatively low firing rates, whereas middle tufted cells responded with higher firing rates. Examination of the molecular receptive range (MRR) indicated that most mitral cells exhibited a robust inhibitory MRR, whereas a majority of middle tufted cells showed no or only a weak inhibitory MRR. In addition, structurally different odorants that activated neighboring clusters inhibited the spike activity of mitral cells, whereas they caused no or only a weak inhibition in the middle tufted cells. Furthermore, responses of mitral cells to an adequate excitatory odorant were greatly inhibited by mixing the odorant with other odorants that activated neighboring glomeruli. In contrast, odorants that activated neighboring glomeruli did not significantly inhibit the responses of middle tufted cells to the adequate excitatory odorant. These results indicate a clear difference between mitral and middle tufted cells in the manner of decoding the glomerular odor maps.     

Side-specific olfactory conditioning leads to more specific odor representation between sides but not within sides in the honeybee antennal lobes

Honeybees can be trained to associate odorants to sucrose reward by conditioning the proboscis extension response. Using this paradigm, we have recently shown that bees can solve a side-specific task: they learn simultaneously to discriminate a reinforced odor A from a non-reinforced odor B at one antenna (A+B−) and the reversed problem at the other antenna (A−B+). Side-specific (A+B−/B+A−) conditioning is an interesting tool to measure neurophysiological changes due to olfactory learning because the same odorant is excitatory (CS+) on one brain side and inhibitory (CS−) on the opposite side. In the bee brain, the antennal lobe (AL) is the first olfactory relay where the olfactory memory is established. Using calcium imaging, we compared odor-evoked activity in the functional units, the glomeruli, of the two ALs, both in naive and conditioned individuals. Each odor evoked a different pattern of glomerular activity, which was symmetrical between sides and highly conserved among naive animals. In conditioned bees, response patterns were overall symmetrical but showed more active glomeruli and topical differences between sides. By representing odor vectors in a virtual olfactory space whose dimensions are the responses of 23 identified glomeruli, we found that distances between odor representations on each brain side were significantly higher in conditioned than in naive bees, but only for CS+ and CS−. However, the distance between CS+ and CS− representations was equal to that of naive individuals. Our work suggests that side-specific conditioning decorrelates odor representations between AL sides but not between CS+ and CS− within one AL.     

Olfactory cortical adaptation facilitates detection of odors against background

Detection and discrimination of odors generally, if not always, occurs against an odorous background. On any given inhalation, olfactory receptor neurons will be activated by features of both the target odorant and features of background stimuli. To identify a target odorant against a background therefore, the olfactory system must be capable of grouping a subset of features into an odor object distinct from the background. Our previous work has suggested that rapid homosynaptic depression of afferents to the anterior piriform cortex (aPCX) contributes to both cortical odor adaptation to prolonged stimulation and habituation of simple odor-evoked behaviors. We hypothesize here that this process may also contribute to figure-ground separation of a target odorant from background stimulation. Single-unit recordings were made from both mitral/tufted cells and aPCX neurons in urethan-anesthetized rats and mice. Single-unit responses to odorant stimuli and their binary mixtures were determined. One of the odorants was randomly selected as the background and presented for 50 s. Forty seconds after the onset of the background stimulus, the second target odorant was presented, producing a binary mixture. The results suggest that mitral/tufted cells continue to respond to the background odorant and, when the target odorant is presented, had response magnitudes similar to that evoked by the binary mixture. In contrast, aPCX neurons filter out the background stimulus while maintaining responses to the target stimulus. Thus the aPCX acts as a filter driven most strongly by changing stimuli, providing a potential mechanism for olfactory figure-ground separation and selective reading of olfactory bulb output.     

Predicting odorant quality perceptions from multidimensional scaling of olfactory bulb glomerular activity patterns

Odorants and their perceptions differ along multiple dimensions, requiring that a critical examination of any putative neural code directly assess the multidimensional nature of the encoding process. Previous work has examined simple, systematic odorant differences that, regardless of coding strategy, would be expected to produce simple, systematic predictions in neural and behavioral responses. In the present study, an odorant identification confusion matrix task that extracts precise quality relationships across odorants was used to determine whether spatially specific glomerular activity patterns predict perceptual quality relationships for odorants that cannot easily be classified a priori along a single chemical dimension. Multidimensional scaling (MDS) analysis of odorant pattern similarity measures derived from the comparison of [14C]-2-deoxyglucose glomerular activity pattern data yielded a two-dimensional odorant activity space that was highly significantly predictive of similarly obtained odorant perceptual spaces, uniformly across animals. These results strongly support the relevance of global spatial patterns in the olfactory bulb to the encoding of odor quality.