Dual functions of mammalian olfactory sensory neurons as odor detectors and mechanical sensors

Most sensory systems are primarily specialized to detect one sensory modality. Here we report that olfactory sensory neurons (OSNs) in the mammalian nose can detect two distinct modalities transmitted by chemical and mechanical stimuli. As revealed by patch-clamp recordings, many OSNs respond not only to odorants, but also to mechanical stimuli delivered by pressure ejections of odor-free Ringer solution. The mechanical responses correlate directly with the pressure intensity and show several properties similar to those induced by odorants, including onset latency, reversal potential and adaptation to repeated stimulation. Blocking adenylyl cyclase or knocking out the cyclic nucleotide-gated channel CNGA2 eliminates the odorant and the mechanical responses, suggesting that both are mediated by a shared cAMP cascade. We further show that this mechanosensitivity enhances the firing frequency of individual neurons when they are weakly stimulated by odorants and most likely drives the rhythmic activity (theta oscillation) in the olfactory bulb to synchronize with respiration.     

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.     

Ciliary neurotrophic factor-immunoreactivity in olfactory sensory neurons

Ciliary neurotrophic factor (CNTF) has been implicated in processes of neuroprotection, axonal regeneration and synaptogenesis in the lesioned CNS. In the olfactory system, which is characterized by particularly robust neuroplasticity throughout life, the concentration of CNTF is high even under physiological conditions. In the present study, the cellular localization of CNTF-immunoreactivity was studied in the rat and mouse olfactory epithelium. In both species, individual olfactory sensory neurons (ONs) displayed intense CNTF-immunoreactivity. The number of CNTF-ir ONs varied interindividually in rats and was lower in mice than in rats. In olfactory epithelia of mice expressing beta-galactosidase under control of the CNTF promoter, cells of the ON layer were immunoreactive for the reporter protein. CNTF-ir ONs were olfactory marker protein-positive and growth associated protein 43-negative. CNTF-ir ONs lacked apoptotic markers, and the number of specifically labeled ONs was apparently unchanged after light chemical lesioning of the epithelium, indicating that CNTF-immunoreactivity was not associated with ON death. Electron microscopy of CNTF-ir ON axons in innervated olfactory bulb glomeruli documented that they formed typical ON axonal synapses with target neurons. Three dimensional reconstructions of bulb pairs showed a striking similarity of the positions of glomeruli innervated by CNTF-ir ON axons in left and right bulbs of individual animals and interindividually. The number of innervated glomeruli differed interindividually in rats and was lower in mice than in rats. The results show that in rodents CNTF-immunoreactivity occurs in a subset of mature, functionally competent ONs. The localization of target glomeruli suggests that CNTF-immunoreactivity may be associated with the expression and/or activation of specific olfactory receptor proteins.     

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.     

Effects of reproductive state on olfactory sensitivity suggest odor specificity

Previous studies of reproductive state and olfactory sensitivity in women have not directly compared thresholds for social and environmental odors. Here, we used successive dilutions presented in a staircase protocol to determine olfactory thresholds for androstadienone, a social odor produced by men, and rose, an environmental odor signaling a source of micronutrients essential for successful implantation, prenatal development and maternal health. Fertile women were more sensitive to the social than the environmental odor, while women using oral contraceptives, a non-fertile hormonal state similar to early pregnancy, were more sensitive to the environmental odor. This preliminary study sets the stage for further work on the interaction between hormonal states and sensitivity to specific odors with reproductive significance.     

Dendritic spikes induced in fast pyramidal tract neurons by thalamic stimulation

Summary In deeply anesthetized cats the synaptic events induced in fast pyramidal tract cells (Pt cells) by ventrolateral (VL) stimulation were analyzed with intracellular recordings. In 40% of the fastest conducting Pt neurons it was found that VL stimulation induced fast depolarizing potentials (FPPs) with or without underlying excitatory postsynaptic potentials (EPSPs). These FPPs were all or none fast rising events lasting about 2 ms. They could be induced by brachium conjunctivum stimulation or they could also occur spontaneously. Those occurring spontaneously had amplitude and time course similar to those evoked by VL stimulation suggesting that they were of thalamic origin. On the basis of their amplitude and lack of collision with antidromic action potentials FPPs were neither axonal nor IS events. Passage of hyperpolarizing currents could block them in an all or none manner. It is concluded that FPPs represent electrotonically attenuated dendritic spikes generated in small side branches (oblique ascending dendrites) or fast Pt neurons where VL terminals most probably establish their synaptic contacts.Supported by the Medical Research Council of Canada (MA 5877)M. Deschênes is a MRC scholar     

Responses of olfactory bulb neurons to repeated odor stimulations in awake freely-breathing rabbits

Thirty-one olfactory bulb neurons were recorded in the olfactory bulbs of unanaesthetized rabbits during repeated stimulations. Their single-unit activity associated with the inspiratory phases of the respiratory cycles and that associated with the expiratory phases were processed separately. When responses were classified into 3 types, i.e., excitation, inhibition and null, it was found that a large number of neurons presented variable responses to repeated stimulations with the same stimulus. However, the passage from one type to another was found to be limited: responses by excitation or inhibition to the first stimulation turned into null responses only; null responses turned into either excitation or inhibition. Inspiration- and expiration-related responses were also subjected to a principal component analysis in order to determine whether changes in responses were compatible with a reliable coding of the qualitative properties of a stimulus. The results indicated that the repeated presentations of an odorant induced fairly similar profiles of activity across the set of neurons while different odorants induced clearly discriminable profiles. It is concluded that repeated stimulations do not blur the characteristic features of the across-neuron profile of response of an odorant in the olfactory bulb despite the variability of the responses of the neurons which compose the profile.     

Dynamics of olfactory bulb input and output activity during odor stimulation in zebrafish

The processing of odor-evoked activity in the olfactory bulb (OB) of zebrafish was studied by extracellular single unit recordings from the input and output neurons, i.e., olfactory receptor neurons (ORNs) and mitral cells (MCs), respectively. A panel of 16 natural amino acid odors was used as stimuli. Responses of MCs, but not ORNs, changed profoundly during the first few hundred milliseconds after response onset. In MCs, but not ORNs, the total evoked excitatory activity in the population was initially odor-dependent but subsequently converged to a common level. Hence, the overall population activity is regulated by network interactions in the OB. The tuning widths of both ORN and MC response profiles were similar and, on average, stable over time. However, when analyzed for individual neurons, MC response profiles could sharpen (excitatory response to fewer odors) or broaden (excitatory response to more odors), whereas ORN response profiles remained nearly unchanged. Several observations indicate that dynamic inhibition plays an important role in this remodeling. Finally, the reliability of odor identification based on MC population activity patterns improved over time, whereas odor identification based on ORN activity patterns was most reliable early in the odor response. These results demonstrate that several properties of MC, but not ORN, activity change during the initial phase of the odor response with important consequences for odor-encoding activity patterns. Furthermore, our data indicate that inhibitory interactions in the OB are important in dynamically shaping the activity of OB output neurons.     

Changes in electrical activity of rabbit olfactory bulb and cortex to conditioned odor stimulation

Rabbits with chronically implanted electrodes in olfactory bulb and cortex were classically conditioned to give an increase in relative frequency of sniffing to odor stimuli (CS+) reinforced with mild electric shock. Electroencephalographic high-frequency (35-85 Hz) bursts were recorded from an ensemble of nine bulbar depth electrodes and a second ensemble of 50 cortical surface electrodes. The olfactory cortex responded to the CS+ with sustained elevation of burst amplitude even though the olfactory bulb, from which it receives its primary centripetal input, underwent a marked decline in burst amplitude during the same time period. The amplitude reduction was not spatially uniform: The burst of the bulbar region that declined most in amplitude had the greatest phase lag with respect to the bulbar ensemble average burst. These effects were learning related because they did not occur for CS+ trials at the beginning of conditioning or for unreinforced control trials at any time.     

Effect of antidromic stimulation of the glossopharyngeal nerve on afferent discharges occurring with and without sensory stimulation of the frog tongue

Repetitive stimulation of the bullfrog glossopharyngeal nerve at a rate of 100 Hz for 10 s and at a supra-maximal intensity resulted in little depression of the glossopharyngeal nerve responses to application of quinine and mechanical taps to the tongue, in a 30-40% decrease in the responses to salt, acid, water and warmed saline and in a 80% decrease in the number of spontaneous discharges from the tongue. Such a selective depression existed throughout changes in the frequency and the duration of the antidromic stimuli. The phenomena were attributed to antidromic impulses in high-threshold glossopharyngeal nerve afferents.     

Facilitatory effect of antidromic stimulation on milk ejection-related activation of oxytocin neurons during suckling in the rat

Oxytocin neurons of the paraventricular nucleus were identified by their antidromic response to stimulation of the neurohypophysis and a short high-frequency burst of spikes displayed before reflex milk ejection in the urethane-anesthetized lactating rat. During suckling, the milk ejection-related bursts recurred at regular intervals. Stimulation of the neurohypophysis at 50-300 pulses/s for 0.5-6 s (current 1 mA) evoked additional burst(s) with the amplitude higher than those of spontaneously occurring ones. Stimulation with subthreshold current for antidromic response or constant-collision test could also facilitate the neurosecretory bursts. Possibly, oxytocin released within the magnocellular nuclei is responsible for these effects.     

Intracellular responses of identified rat olfactory bulb interneurons to electrical and odor stimulation

1. Intracellular recordings were made from 28 granule cells and 6 periglomerular cells of the rat olfactory bulb during odor stimulation and electrical stimulation of the olfactory nerve layer (ONL) and lateral olfactory tract (LOT). Neurons were identified by injection of horseradish peroxidase (HRP) or biocytin and/or intracellular response characteristics. Odorants were presented in a cyclic sniff paradigm, as reported previously. 2. All interneurons could be activated from a wide number of stimulation sites on the ONL, with distances exceeding their known dendritic spreads and the dispersion of nerve fibers within the ONL, indicating that multisynaptic pathways must also exist at the glomerular region. All types of interneurons also responded to odorant stimulation, showing a variety of responses. 3. Granule cells responded to electrical stimulation of the LOT and ONL as reported previously. However, intracellular potential, excitability, and conductance analysis suggested that the mitral cell-mediated excitatory postsynaptic potential (EPSP) is followed by a long inhibitory postsynaptic potential (IPSP). An early negative potential, before the EPSP, was also observed in every granule cell and correlated with component I of the extracellular LOT-induced field potential. We have interpreted this negativity as a "field effect," that may be diagnostic of granule cells. 4. Most granule cells exhibited excitatory responses to odorant stimulation. Odors could produce spiking responses that were either nonhabituating (response to every sniff) or rapidly habituating (response to first sniff only). Other granule cells, while spiking to electrical stimulation, showed depolarizations that did not evoke spikes to odor stimulation. These depolarizations were transient with each sniff or sustained across a series of sniffs. These physiological differences to odor stimulation correlated with granule cell position beneath the mitral cell layer for 12 cells, suggesting that morphological subtypes of granule cells may show physiological differences. Some features of the granule cell odor responses seem to correlate with some of the features we have observed in mitral/tufted cell intracellular recordings. Only one cell showed inhibition to odors. 5. Periglomerular (PG) cells showed a response to ONL stimulation that was unlike that found in other olfactory bulb neurons. There was a long-duration hyperpolarization after a spike and large depolarization or burst of spikes (20-30 ms in duration). Odor stimulation produced simple bursts of action potentials, Odor stimulation produced simple bursts of action potentials, suggesting that PG cells may simply follow input from the olfactory nerve.(ABSTRACT TRUNCATED AT 400 WORDS)     

Processing of bile salt odor information by single olfactory bulb neurons in the channel catfish

A chemotopic map of biologically relevant odorants (that include amino acids, bile salts, and nucleotides) exists in the olfactory bulb (OB) of channel catfish, Ictalurus punctatus. Neurons processing bile salt odorant information lie medially within this OB map; however, information as to how single neurons process bile salt odorant information is lacking. In the present report, recordings were obtained from 51 OB neurons from 30 channel catfish to determine the excitatory molecular receptive range (EMRR) of bile salt responsive neurons. All recordings were performed in vivo within the medial portions of the OB using extracellular electrophysiological techniques. Excitatory thresholds to bile salts typically ranged between 0.1 and 10 muM. The bile salt specificity of OB neurons were divided into three groups: neurons excited by taurine-conjugated bile salts only (group T), neurons excited by nonconjugated bile salts only (group N), and neurons excited by at least one member of each of the three classes of bile salts tested (group G). In addition to the conjugating group at C24 of the side-chain, OB neurons discriminated bile salts by the molecular features present at three other carbon positions (C3, C7, and C12) along the steroid backbone. These data suggest that OB neurons are selectively excited by combinations of molecular features found on the side-chain and along the steroid nucleus of bile salt molecules.     

Calcium-activated chloride currents in olfactory sensory neurons from mice lacking bestrophin-2

Olfactory sensory neurons use a chloride-based signal amplification mechanism to detect odorants. The binding of odorants to receptors in the cilia of olfactory sensory neurons activates a transduction cascade that involves the opening of cyclic nucleotide-gated channels and the entry of Ca²⁺ into the cilia. Ca²⁺ activates a Cl⁻ current that produces an efflux of Cl⁻ ions and amplifies the depolarization. The molecular identity of Ca²⁺-activated Cl⁻ channels is still elusive, although some bestrophins have been shown to function as Ca²⁺-activated Cl⁻ channels when expressed in heterologous systems. In the olfactory epithelium, bestrophin-2 (Best2) has been indicated as a candidate for being a molecular component of the olfactory Ca²⁺-activated Cl⁻ channel. In this study, we have analysed mice lacking Best2. We compared the electrophysiological responses of the olfactory epithelium to odorant stimulation, as well as the properties of Ca²⁺-activated Cl⁻ currents in wild-type (WT) and knockout (KO) mice for Best2. Our results confirm that Best2 is expressed in the cilia of olfactory sensory neurons, while odorant responses and Ca²⁺-activated Cl⁻ currents were not significantly different between WT and KO mice. Thus, Best2 does not appear to be the main molecular component of the olfactory channel. Further studies are required to determine the function of Best2 in the cilia of olfactory sensory neurons.     

Cyclic AMP cascade mediates the inhibitory odor response of isolated toad olfactory receptor neurons

Odor stimulation may excite or inhibit olfactory receptor neurons (ORNs). It is well established that the excitatory response involves a cyclic AMP (cAMP) transduction mechanism that activates a nonselective cationic cyclic nucleotide-gated (CNG) conductance, accompanied by the activation of a Ca2+-dependent Cl(-) conductance, both causing a depolarizing receptor potential. In contrast, odor inhibition is attributed to a hyperpolarizing receptor potential. It has been proposed that a Ca2+-dependent K+ (K(Ca)) conductance plays a key role in odor inhibition, both in toad and rat isolated olfactory neurons. The mechanism underlying odor inhibition has remained elusive. We assessed its study using various pharmacological agents and caged compounds for cAMP, Ca2+, and inositol 1,4,5-triphosphate (InsP3) on isolated toad ORNs. The odor-triggered K(Ca) current was reduced on exposing the cell either to the CNG channel blocker LY83583 (20 microM) or to the adenylyl cyclase inhibitor SQ22536 (100 microM). Photorelease of caged Ca2+ activated a Cl- current sensitive to niflumic acid (10 microM) and a K+ current blockable by charybdotoxin (20 nM) and iberiotoxin (20 nM). In contrast, photoreleased Ca2+ had no effect on cells missing their cilia, indicating that these conductances are confined to the cilia. Photorelease of cAMP induced a charybdotoxin-sensitive K+ current in intact ORNs. Photorelease of InsP3 did not increase the membrane conductance of olfactory neurons, arguing against a direct role of InsP3 in chemotransduction. We conclude that a cAMP cascade mediates the activation of the ciliary Ca2+-dependent K+ current and that the Ca2+ ions that activate the inhibitory current enter the cilia through CNG channels.     

Cholinergic microvillous cells in the mouse main olfactory epithelium and effect of acetylcholine on olfactory sensory neurons and supporting cells

The mammalian olfactory epithelium is made up of ciliated olfactory sensory neurons (OSNs), supporting cells, basal cells, and microvillous cells. Previously, we reported that a population of nonneuronal microvillous cells expresses transient receptor potential channel M5 (TRPM5). Using transgenic mice and immunocytochemical labeling, we identify that these cells are cholinergic, expressing the signature markers of choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter. This result suggests that acetylcholine (ACh) can be synthesized and released locally to modulate activities of neighboring supporting cells and OSNs. In Ca(2+) imaging experiments, ACh induced increases in intracellular Ca(2+) levels in 78% of isolated supporting cells tested in a concentration-dependent manner. Atropine, a muscarinic ACh receptor (mAChR) antagonist suppressed the ACh responses. In contrast, ACh did not induce or potentiate Ca(2+) increases in OSNs. Instead ACh suppressed the Ca(2+) increases induced by the adenylyl cyclase activator forskolin in some OSNs. Supporting these results, we found differential expression of mAChR subtypes in supporting cells and OSNs using subtype-specific antibodies against M(1) through M(5) mAChRs. Furthermore, we found that various chemicals, bacterial lysate, and cold saline induced Ca(2+) increases in TRPM5/ChAT-expressing microvillous cells. Taken together, our data suggest that TRPM5/ChAT-expressing microvillous cells react to certain chemical or thermal stimuli and release ACh to modulate activities of neighboring supporting cells and OSNs via mAChRs. Our studies reveal an intrinsic and potentially potent mechanism linking external stimulation to cholinergic modulation of activities in the olfactory epithelium.     

Conditional ablation of mature olfactory sensory neurons mediated by diphtheria toxin receptor

The vertebrate olfactory epithelium provides an excellent model system to study the regulatory mechanisms of neurogenesis and neuronal differentiation due to its unique ability to generate new sensory neurons throughout life. The replacement of olfactory sensory neurons is stimulated when damage occurs in the olfactory epithelium. In this study, transgenic mice, with a transgene containing human diphtheria toxin receptor under the control of the olfactory marker protein promoter (OMP-DTR), were generated in which the mature olfactory sensory neurons could be specifically ablated when exposed to diphtheria toxin. Following diphtheria toxin induced neuronal ablation, we observed increased numbers of newly generated growth associated protein 43 (GAP43)-positive immature olfactory sensory neurons. OMP-positive neurons were continuously produced from the newly generated GAP43-positive cells. The expression of the signal transduction components adenylyl cyclase type III and the G-protein α subunit G_{α olf} was sensitive to diphtheria toxin exposure and their levels decreased dramatically preceding the disappearance of the OMP-positive sensory neurons. These data validate the hypothesis that OMP-DTR mice can be used as a tool to ablate the mature olfactory sensory neurons in a controlled fashion and to study the regulatory mechanisms of the neuronal replacement.