State-dependent sculpting of olfactory sensory neurons is attributed to sensory enrichment,odor deprivation,and aging

Gene-targeted deletion of the predominant Shaker potassium channel, Kv1.3, in the mitral cells of the olfactory bulb, decreases the number of presynaptic, odorant receptor (OR)-identified olfactory sensory neurons (OSNs) in the main olfactory epithelium (MOE) and alters the nature of their postsynaptic connections to mitral cell targets. The current study examined whether OSN density was state-dependent by examining the impact of (1) odor enrichment, (2) sensory deprivation, and (3) aging upon the number of P2- or M72-expressing neurons. Histological approaches were used to quantify the number of OSNs across entire epithelia for wildtype (WT) vs. Kv1.3-null (KO) mice bred onto an ORtauLacZ reporter background. Following either odor enrichment or early unilateral naris-occlusion, the number of M72-expressing OSNs was significantly decreased in WT mice, but was unchanged in KO animals. Following naris-occlusion, the number of P2-expressing OSNs was decreased regardless of genotype. Animals that were reared to 2 years of age demonstrated loss of both P2- and M72-expressing OSNs in WT mice and a concomitant loss of only M72-expressing neurons in KO mice. These findings suggest that voltage-gated activity of the mitral cells is important for OSN plasticity, and can prevent neuronal loss via sensory- and OR-dependent mechanisms.     

Olfactory sensitivity, learning and cognition in young adult and aged male Wistar rats

Psychophysical experiments with male Wistar rats, ranging from 2 to more than 25 months old, revealed age-related differences in olfactory sensitivity. The highest sensitivities were found in rats 13 months old, and the lowest sensitivity was found in the group aged 25 months and older. Consequently, we considered the hypotheses that young rats will require less time and less trials than aged conspecifics to learn an olfactory discrimination task and that olfactory cognitive abilities will be reduced in older individuals. Rats were initially trained in an olfactometer using operant techniques to discriminate between the odor ethyl acetate (EA) and clean air. Next, young adult and 28-month-old rats were tested on seven different go/no-go odor discrimination tasks. Aged rats performed as well as young adults did on all tasks and we conclude that, for a variety of odor discrimination problems, aged rats show no deterioration in learning ability. This is the first report on olfactory sensitivity, learning ability and cognition in Wistar rats that have passed the normal life span for this strain. Data show that the inability to learn and cognitive deficits do not necessarily develop with age.     

The Na(+)/Ca(2+) exchanger NCKX4 governs termination and adaptation of the mammalian olfactory response

Sensory perception requires accurate encoding of stimulus information by sensory receptor cells. We identified NCKX4, a potassium-dependent Na(+)/Ca(2+) exchanger, as being necessary for rapid response termination and proper adaptation of vertebrate olfactory sensory neurons (OSNs). Nckx4(-/-) (also known as Slc24a4) mouse OSNs displayed substantially prolonged responses and stronger adaptation. Single-cell electrophysiological analyses revealed that the majority of Na(+)-dependent Ca(2+) exchange in OSNs relevant to sensory transduction is a result of NCKX4 and that Nckx4(-/-) mouse OSNs are deficient in encoding action potentials on repeated stimulation. Olfactory-specific Nckx4(-/-) mice had lower body weights and a reduced ability to locate an odorous source. These results establish the role of NCKX4 in shaping olfactory responses and suggest that rapid response termination and proper adaptation of peripheral sensory receptor cells tune the sensory system for optimal perception.     

Ontogeny of olfaction: Development of the rats' sensitivity to urine and amyl acetate

Rat pups, 1 to 17 days of age, were tested for sensitivity to two olfactants, amyl acetate and adult rat urine. Biological and non-biological olfactory stimuli were generated by sparging and delivered to subjects via a dilution olfactometer. Unconditioned respiratory responses, odor-induced polypnea and sniffing, were used to measure detection of an odorant injected into a background stream of filtered air. Amyl acetate was presented in an ascending series of concentrations. Pups of all ages detected amyl acetate; chemosensitivity increased with age to all 3 concentrations of this nonbiological stimulus. Two concentrations of adult rat urine odor were equated to the strength of amyl acetate for 9-day-olds. Each normalized urine stimulus was then tested across the full range of age groups. Again, there was a dramatic age-related increase in chemosensitivity. Moreover, there was no indication of differential sensitivity to these biological and non-biological olfactants. These data were discussed within methodological and conceptual frameworks related to analyses of early olfactory and behavioral ontogenesis.     

Odor specificity of habituation in the rat anterior piriform cortex

Exposure to odorants results in a rapid (<10 s) reduction in odor-evoked activity in the rat piriform cortex despite relatively maintained afferent input from olfactory bulb mitral cells. To further understand this form of cortical plasticity, a detailed analysis of its odor specificity was performed. Habituation of odor responses in anterior piriform cortex single units was examined in anesthetized, freely breathing rats. The magnitude of single-unit responses of layer II/III neurons to 2-s odor pulses were examined before and after a 50-s habituating stimulus of either the same or different odor. The results demonstrated that odor habituation was odor specific, with no significant cross-habituation between either markedly different single odors or between odors within a series of straight chain alkanes. Furthermore, habituation to binary 1:1 mixtures produced minimal cross-habituation to the components of that mixture. These latter results may suggest synthetic odor processing in the olfactory system, with novel odor mixtures processed as unique stimuli. Potential mechanisms of odor habituation in the piriform cortex must be able to account for the high degree of specificity of this effect.     

Field potential response changes in the rabbit olfactory bulb accompany behavioral habituation during the repeated presentation of unreinforced odors

Summary Experiments were performed on waking rabbits to investigate the changes in both sniffing behavior and local field potential responses in the olfactory bulb during repeated exposure to unreinforced odors. Six rabbits were each implanted with 2 pairs of electrodes for differential recording of the bulbar extracellular field potential. Each animal was given 3 sequential sessions to each of 2 separate odors on 6 consecutive days, while monitoring the bulbar field potential activity and sniffing behavior. Behavioral sniffing responses exhibited rapid within-session decrement in amplitude and long term decrement across sessions. The within-session decrement showed spontaneous recovery between sessions. Both decremental changes in sniffing behavior were accompanied by changes in the bulbar field potential responses. The responses to novel odors were characterized by a reduction in amplitude of high frequency activity (40–80 Hz) and a corresponding increase in amplitude of low frequency activity (15–25 Hz). The high frequency component of the responses showed an initial increase in frequency to a novel odor on the first 3 presentations followed by a rapid decrease in frequency on subsequent trials in the first session which stabilized thereafter. No change in frequency or relative amplitude was observed for the low frequency component. The absolute difference between the odor evoked activity and the preceeding control activity measured on each trial showed a significant decrement across sessions with no evidence for spontaneous recovery. The results demonstrate that olfactory bulb responses to novel unreinforced odors show both rapid and long-term changes which parallel changes in sniffing behavior. These changes, which have been predicted by a theoretical model of the olfactory bulb (Freeman 1979a, b), are postulated to reduce the spatial specificity of the response pattern to unreinforced odors.     

The impact of olfactory dysfunction on interoceptive awareness

This study aimed to investigate how the impairment of the olfactory system influences interoception. Interoception is known as the awareness of one's body or the sense of the condition of the body; more precisely, this construct is defined as the processing, representation, and perception of the internal physical state. Interoceptive sensitivity and chemosensory performance were assessed in 77 subjects, including 43 functional anosmics, 18 hyposmics, and 16 healthy controls. Interoceptive awareness was predicted by odor detection threshold, as well as the duration of olfactory loss in patients who suffered from reduced olfactory function—the longer the olfactory impairment, the worse the perception of bodily signals. The results of this study will significantly contribute to the basic understanding of the multifaceted effects of olfactory alterations.     

Olfactory sensitivity of rats reared in an odorous or deodorized environment

Male rats were exposed from the age of 1 or 14 days to the odor of cyclohexanone for two months. Four concentrations of cyclohexanone were used, ranging from near threshold to approximately 30,000 times this level. Other rats were reared in an environment of deodorized air or in a normal rat colony. The olfactory sensitivity of all rats for cyclohexanone was determined using an air dilution olfactometer and a two-choice (odor vs air) test procedure. There were no differences in the learning rates of groups reared in different environments for the detection task and the acuity of rats for cyclohexanone was not altered by exposure to it. Howerver, rats raised in the deodorized air environment were deficient in their ability to detect cyclohexanone. These results support the earlier proposal that rats do not lose sensitivity for an odor following prolonged exposure to it whilst deprivation can lead to a decrease in olfactory sensitivity.     

Age-related loss of olfactory sensitivity: Association to dopamine transporter binding in putamen

The relationship between age-related reductions in the binding potential for the striatal dopamine transporter (DAT) and age-related deficits in olfactory sensitivity was examined in 12 subjects ranging from 36 to 82 years of age. Positron emission tomography (PET) and the radioligand [¹¹C]β-CIT-FE were used to determine DAT binding in two striatal regions, the caudate and the putamen. The results showed age-related losses of DAT binding from early to late adulthood of similar size for caudate and putamen, and there was a pronounced age deterioration in olfactory sensitivity. Importantly, the age-related olfactory deficit was associated with reductions in DAT binding in putamen, but not caudate. Also, DAT binding in putamen added systematic variance in odor threshold after controlling for age. The findings indicate that DAT binding in putamen is related to age-related olfactory deficits, as well as to odor sensitivity independently of age.     

Olfactory dysfunction affects thresholds to trigeminal chemosensory sensations

Next to olfaction and gustation, the trigeminal system represents a third chemosensory system. These senses are interconnected; a loss of olfactory function also leads to a reduced sensitivity in the trigeminal chemosensory system. However, most studies so far focused on comparing trigeminal sensitivity to suprathreshold stimuli; much less data is available with regard to trigeminal sensitivity in the perithreshold range. Therefore we assessed detection thresholds for CO₂, a relatively pure trigeminal stimulus in controls and in patients with olfactory dysfunction (OD). We could show that OD patients exhibit higher detection thresholds than controls. In addition, we were able to explore the effects of different etiologies of smell loss on trigeminal detection thresholds. We could show that in younger subjects, patients suffering from olfactory loss due to head trauma are more severely impaired with regard to their trigeminal sensitivity than patients with isolated congenital anosmia. In older patients, we could not observe any differences between different etiologies, probably due to the well known age-related decrease of trigeminal sensitivity. Furthermore we could show that a betterment of the OD was accompanied by decreased thresholds. This was most evident in patients with postviral OD. In conclusion, factors such as age, olfactory status and etiology of olfactory disorder can affect responsiveness to perithreshold trigeminal chemosensory stimuli.     

A Histopathological Study on Senile Changes in the Human Olfactory Bulb

Senile changes in the human olfactory bulb were studied histopathologically in 133 individuals ranging in age from 40 to 91 years with a mean age of 64.3 years. Neurofibrillary tangles (NFTs) in the olfactory bulb were observed in 47 subjects aged over 50 years. The frequency of NFTs was 35.3% (47/133) in total and 40.5% (47/116) among subjects 50 years of age or older. The frequency of individuals with NFTs increased linearly with age. Senile plaques (SPs) were observed in 5 individuals over 60 years old at a frequency of 3.8% (5/133) in total and 5.7% (5/88) among individuals 60 years old or more. Granulovacuolar degeneration was rarely seen, and amyloid angiopathy was not recognized. Senile changes in the hippocampus and parahippocampal gyrus were also examined. In some cases, the frequencies of NFTs and SPs in the hippocampal region were not identical to those in the olfactory bulb. However, it was not apparent whether senile changes first appear in the olfactory bulb or in the hippocampal region.     

Discrimination of odors from stressed rats by non-stressed rats

Comparison of earlier reports of rat stress odors is complicated by the many differences in experimental parameters and responses measured. To evaluate whether these stress odors provide a special signal, rats were subjected to different levels of stressful foot-shock in one half of a simple two-compartment test box whilst the other half was clean and unoccupied. The results show that whilst test subjects preferred the half containing odors from non-stressed rats, this preference was decreased by the presence of stress odors to an extent concordant with the level of stressor applied to the odor donors. There were no differences in plasma corticosterone among the odor donors indicating that this hormone is probably not the source of stress odors. Plasma corticosterone levels of the subjects were similar to each other and to the odor donors. Compared to odors from non-stressed rats, stress odors increased the activity of the subjects. The evidence strongly suggests a special signalling function for stress odors although responses to this signal are not stereotyped.     

Cerebellar direct current stimulation enhances motor learning in older adults

Developing novel approaches to combat age related declines in motor function is key to maintaining health and function in older adults, a subgroup of the population that is rapidly growing. Motor adaptation, a form of motor learning, has been shown to be impaired in healthy older subjects compared with their younger counterparts. Here, we tested whether excitatory anodal transcranial direct current stimulation (tDCS) over the cerebellum could enhance adaptation in older subjects. Participants performed a “center-out” reaching task, adapting to the sudden introduction of a visual cursor rotation. Older participants receiving sham tDCS (mean age 56.3 ± 6.8 years) were slower to adapt than younger participants (mean age 20.7 ± 2.1 years). In contrast, older participants who received anodal tDCS (mean age 59.6 ± 8.1 years) adapted faster, with a rate that was similar to younger subjects. We conclude that cerebellar anodal tDCS enhances motor adaptation in older individuals. Our results highlight the efficacy of the novel approach of using cerebellar tDCS to combat age related deficits in motor learning.     

Non-specific olfactory aversion induced by intrabulbar infusion of the GABA(A) receptor antagonist bicuculline in young rats

On postnatal day 12, young rats show an aversion to an odor to which they had been exposed along with presentations of foot shock on postnatal day 11. The acquisition of this aversive learning involves and requires disinhibition of the mitral/tufted cells induced by centrifugal noradrenergic activation during somatosensory stimulation. This olfactory learning is established only for the odor to which the rat has been exposed during conditioning. Infusion of the GABA(A) receptor antagonist bicuculline at a high dose (2.0 nmol/each olfactory bulb) into the olfactory bulb in the presence of an odor is capable of developing olfactory aversive responses without somatosensory stimulation in young rats. The purpose of this study is to characterize the properties of bicuculline-induced aversive responses. In contrast to the odor specificity of aversive learning produced by odor-shock conditioning, bicuculline-induced aversive responses lack odor specificity. Namely, bicuculline infusion in the presence of a citral odor results, in a dose-dependent manner, in subsequent aversive responses to strange odors (benzaldehyde and vanillin) that have never been presented. Moreover, bicuculline infusion alone is sufficient to produce dose-dependent aversive responses to strange odors (citral, benzaldehyde and geraniol).From these results we suggest that disinhibition of mitral/tufted cells from granule cells by bicuculline infusion makes young rats aversive to strange odors non-specifically, as if the rats had learned the odor aversion as a result of odor exposure paired with foot shock. Different mechanisms of disinhibition of the mitral/tufted cells may underlie both the pharmacological manipulation and noradrenergic activation by somatosensory stimulation.     

Selective attention and the perceptual analysis of odor mixtures.

Two psychophysical methods were used to investigate the capacity of humans to identify the constituents of odor mixtures consisting of up to six components. With one method subjects were required to identify all the components present in each stimulus; with the other, a selective attention procedure was used where subjects had to identify only one component at each trial. Little difference was found between the levels of identification obtained with both methods, reinforcing the finding that humans have great difficulty in identifying more than three components in an odor mixture and indicating that it is unlikely that olfactory adaptation influenced the identification process.     

Intramodal blocking between olfactory stimuli in rats

The means by which olfactory systems enable the consistent recognition of biologically meaningful odors that may vary in composition over time is a central problem in olfaction. Experiments in honeybees have suggested a solution to one aspect of this problem by demonstrating that the learning of components in an odor mixture suppresses subsequent learning about additional components that are later added to that mixture. We here show, in rats, that intramodal olfactory blocking is also exhibited in vertebrates, and furthermore that the specific characteristics of this blocking are similar to those observed in intermodal blocking studies. Using an olfactory digging task, we submitted two groups of rats to the following olfactory training protocol: one group was first trained (five trials) on an odor (A), followed by five training trials on a binary mixture (A+B), while the second group was similarly trained on a different odor (C) followed by the binary mixture (A+B). After training, both groups' unrewarded responses to the mixture (A+B), odor (B), and an unconditioned control odor (X) were measured. For four out of five odor sets used in this experiment, a significant difference in digging time was observed between the responses of the two groups to odor B (P<.01), but not between their responses to the binary mixture (A+B) or to the control odor (X). These results demonstrate that pretraining rats with an odorant blocks subsequent learning about another odorant when the latter is presented simultaneously with the pretrained odorant, indicating that the components of a mixture are at least partially perceived as independent elements of this mixture.     

Mitochondrial Ca(2+) mobilization is a key element in olfactory signaling

In olfactory sensory neurons (OSNs), cytosolic Ca(2+) controls the gain and sensitivity of olfactory signaling. Important components of the molecular machinery that orchestrates OSN Ca(2+) dynamics have been described, but key details are still missing. Here, we demonstrate a critical physiological role of mitochondrial Ca(2+) mobilization in mouse OSNs. Combining a new mitochondrial Ca(2+) imaging approach with patch-clamp recordings, organelle mobility assays and ultrastructural analyses, our study identifies mitochondria as key determinants of olfactory signaling. We show that mitochondrial Ca(2+) mobilization during sensory stimulation shapes the cytosolic Ca(2+) response profile in OSNs, ensures a broad dynamic response range and maintains sensitivity of the spike generation machinery. When mitochondrial function is impaired, olfactory neurons function as simple stimulus detectors rather than as intensity encoders. Moreover, we describe activity-dependent recruitment of mitochondria to olfactory knobs, a mechanism that provides a context-dependent tool for OSNs to maintain cellular homeostasis and signaling integrity.     

Compensatory responses to age-related decline in odor quality acuity: Cholinergic neuromodulation and olfactory enrichment

The perceptual differentiation of odors can be measured behaviorally using generalization gradients. The steepness of these gradients defines a form of olfactory acuity for odor quality that depends on neural circuitry within the olfactory bulb and is regulated by cholinergic activity therein as well as by associative learning. Using this system as a reduced model for age-related cognitive decline, we show that aged mice, while maintaining almost the same baseline behavioral performance as younger mice, are insensitive to the effects of acutely elevated acetylcholine, which sharpens generalization gradients in young adult mice. Moreover, older mice exhibit evidence of chronically elevated acetylcholine levels in the olfactory bulb, suggesting that their insensitivity to further elevated levels of acetylcholine may arise because the maximum capacity of the system to respond to acetylcholine has already been reached. We propose a model in which an underlying, age-related, progressive deficit is mitigated by a compensatory cholinergic feedback loop that acts to retard the behavioral effects of what would otherwise be a substantial age-related decline in olfactory plasticity.We also treated mice with 10-day regimens of olfactory environmental enrichment and/or repeated systemic injections of the acetylcholinesterase inhibitor physostigmine. Each treatment alone sharpened odor quality acuity, but administering both treatments together had no greater effect than either alone. Age was not a significant main effect in this study, suggesting that some capacity for acetylcholine-dependent plasticity is still present in aged mice despite their sharply reduced ability to respond to acute increases in acetylcholine levels.These results suggest a dynamical framework for understanding age-related decline in neural circuit processing in which the direct effects of aging can be mitigated, at least temporarily, by systemic compensatory responses. In particular, a decline in cholinergic efficacy can precede any breakdown in cholinergic production, which may help explain the limited effectiveness of cholinergic replacement therapies in combating cognitive decline.     

Reduction of odor and nasal pungency associated with aging

—Aging can seriously blunt suprathreshold sensations mediated by the olfactory receptor system and by common chemical receptors. Despite large individual differences, on the average any given stimulus seemed only about half as intense to the elderly (20 subjects, 65–83 yrs) as to the young (20 subjects, 18–25 yrs). The nature of the loss was a constant percentage reduction of perceived magnitude at stimulus levels from weak to strong. The stimuli were iso-amyl butyrate (a nonirritating fruity odor) and CO₂ (which is practically odorless but triggers common chemical sensations effectively). The method used was magnitude matching, by which subjects made numerical estimates of the perceived magnitude of various levels of the two chemical stimuli and of the loudness of low-pitched noises. The loudness estimates served to adjust each subject's chemical estimates to help compensate for individual idiosyncrasies in the use of numbers and potential biases associated with age. Common chemical and olfactory losses seem to be unrelated; aging can dull one sense and leave the other acute.     

Consolidation and expression of a shock-induced odor preference in rat pups is facilitated by opioids

To support nipple attachment and huddling, rat pups must learn to approach and prefer maternal odor. Similar to other altricial species, rat pups have a sensitive period for learning this odor preference, which ends around postnatal day (PN) 10 and coincides with the emergence of walking. One characteristic of this sensitive period is that an odor paired with moderate shock elicits an odor preference. After PN10, this behavioral training produces an odor aversion, although pain threshold remains unchanged. Recently, we demonstrated that the endogenous opioid system might be a key element in the acquisition of the shock-induced odor preference during the sensitive period since antagonism of this system disrupts odor preference learning. In older pups, acquisition of a shock-induced odor aversion was unaffected by opioid system manipulation. The purpose of these experiments was to further elucidate the role of opioids in infant olfactory learning through assessment of memory consolidation and expression during and after the sensitive period. In Experiment 1, we demonstrate that naltrexone (NTX), a nonspecific opioid antagonist, given immediately following odor-shock conditioning during the sensitive period, blocks odor preference formation and yields an odor aversion. However, the same treatment does not disrupt consolidation of an odor aversion in older pups. In Experiment 2, we demonstrate that during the sensitive period, NTX disrupts expression of the shock-induced odor preference, but not the learned odor aversion in older pups. Results using this model of attachment suggest that opioids have an important role in the acquisition, consolidation, and expression of early olfactory preferences. Furthermore, since prenatal drug exposure is known to alter the endogenous opioid system, these results highlight the capacity of prenatal opiate exposure to disrupt early infant learning and attachment.