Preparation of isolated mouse olfactory receptor neurons

A method is described for producing large numbers of isolated olfactory receptor neurons from adult mouse nasal epithelium. The dissociated neurons and other cell types isolated from nasal epithelium retain their morphology and can be identified visually. The neurons were judged to be intact and viable by trypan blue dye exclusion, the presence of olfactory marker protein (OMP), and a variety of electrophysiological measurements indicating the presence of substantial membrane potentials, low levels of intracellular Ca²⁺, and the ability to fire action potentials. The receptor neurons and other cell types produced by this method are amenable to the patch-clamp technique and to immunohistochemical studies.     

Intracellular recordings from isolated salamander olfactory receptor neurons

Isolated receptor cells were obtained by enzymatic dissociation of nasal sacs from the land-phase tiger salamander. The isolated cells have an ovoid soma, a dendrite of variable length which terminates in a cilia-bearing knob and an axon, also of variable length. Intracellular recordings were obtained using patch pipettes. Good recordings were characterized by resting potentials of -40 mV, high input impedance and the presence of fast overshooting action potentials upon depolarization or rebound excitation. With one cell, chemical stimulation evoked large depolarizations which produced action potentials. The reversal potential of this response was +2.7 mV. The results show that these cells can be dissociated for patch recordings, and they support previous studies indicating that transduction of olfactory stimuli leads to a depolarization of vertebrate olfactory receptor neurons.     

Cyclic AMP mediates the activity of steroid hormones

There are two classes of hormones, those derived from amino acids and those with a steroid nucleus. Those from amino acids are said to act by raising intra-cellular levels of cyclic AMP. Because the glucocorticoids (and, in general, all steroid hormones) do not raise tissue levels of cyclic AMP, their activity is not considered to be mediated by this nucleotide. I hypothesize that the role of glucocorticoids is to sensitize the cell to the effector molecule, cyclic AMP. This concept is founded on the following, oft-repeated observations from the literature:- Cyclic AMP can always mimic glucocorticoid activity. Tissues from adrenalectomized animals show greatly diminished sensitivity to cyclic AMP. Hormones which act by elevating cyclic AMP levels, and exogenously added cyclic AMP, exert their effect only at levels much higher than are required in normal tissue. If the ability of cyclic AMP and glucocorticoids to initiate a particular activity is compared, cyclic AMP is the more rapid effector. A system maximally stimulated by cyclic AMP is but slightly enhanced by the addition of a glucocorticoid, whereas a system maximally stimulated by glucocorticoids is strongly stimulated by adding cyclic AMP.     

Cyclic nucleotide-activated currents in cultured olfactory receptor neurons of the hawkmoth Manduca sexta

Moth pheromones cause rises in intracellular Ca(2+) concentrations that activate Ca(2+)-dependent cation channels in antennal olfactory receptor neurons. In addition, mechanisms of adaptation and sensitization depend on changes in cyclic nucleotide concentrations. Here, cyclic nucleotide-activated currents in cultured olfactory receptor neurons of the moth Manduca sexta are described, which share properties with currents through vertebrate cyclic nucleotide-gated channels. The cyclic nucleotide-activated currents of M. sexta carried Ca(2+) and monovalent cations. They were directly activated by cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), modulated by Ca(2+)/calmodulin, and inhibited by lanthanum. M. sexta cyclic nucleotide-activated currents developed in an all-or-none manner, which suggests that the underlying channels are coupled and act coordinately. At least one cAMP- and two cGMP-activated nonselective cation currents could be distinguished. Compared with the cAMP-activated current, both cGMP-activated currents appeared to conduct more Ca(2+) and showed a stronger down-regulation by Ca(2+)/calmodulin-dependent negative feedback. Furthermore, both cGMP-activated currents differed in their Ca(2+)-dependent inhibition. Thus M. sexta olfactory receptor neurons, like vertebrate sensory neurons, appear to express nonselective cyclic nucleotide-activated cation channels with different subunit compositions. Besides the nonselective cyclic nucleotide-activated cation currents, olfactory receptor neurons express a cAMP-dependent current. This current resembled a protein kinase-modulated low-voltage-activated Ca(2+) current.     

Effects of odor stimulation on antidromic spikes in olfactory sensory neurons

Spikes were evoked in rat olfactory sensory neuron (OSN) populations by electrical stimulation of the olfactory bulb nerve layer in pentobarbital anesthetized rats. The latencies and recording positions for these compound spikes showed that they originated in olfactory epithelium. Dual simultaneous recordings indicated conduction velocities in the C-fiber range, around 0.5 m/s. These spikes are concluded to arise from antidromically activated olfactory sensory neurons. Electrical stimulation at 5 Hz was used to track changes in the size and latency of the antidromic compound population spike during the odor response. Strong odorant stimuli suppressed the spike size and prolonged its latency. The latency was prolonged throughout long odor stimuli, indicating continued activation of olfactory receptor neuron axons. The amounts of spike suppression and latency change were strongly correlated with the electroolfactogram (EOG) peak size evoked at the same site across odorants and across stimulus intensities. We conclude that the curve of antidromic spike suppression gives a reasonable representation of spiking activity in olfactory sensory neurons driven by odorants and that the correlation of peak spike suppression with the peak EOG shows the accuracy of the EOG as an estimate of intracellular potential in the population of olfactory sensory neurons. In addition, these results have important implications about traffic in olfactory nerve bundles. We did not observe multiple peaks corresponding to stimulated and unstimulated receptor neurons. This suggests synchronization of spikes in olfactory nerve, perhaps by ephaptic interactions. The long-lasting effect on spike latency shows that action potentials continue in the nerve throughout the duration of an odor stimulus in spite of many reports of depolarization block in olfactory receptor neuron cell bodies. Finally, strong odor stimulation caused almost complete block of antidromic spikes. This indicates that a very large proportion of olfactory axons was activated by single strong odor stimuli.     

3-Nitrotyrosine immunoreactivity in olfactory receptor neurons of patients with Alzheimer's disease: implications for impaired odor sensitivity

Olfactory sensory function is impaired in patients with the diagnosis of probable Alzheimer's disease (AD) compared to elderly controls, and the olfactory epithelium (OE) of AD patients exhibits several pathological changes characteristic of the AD brain. To confirm that the populations from whom our postmortem tissues are obtained exhibit similar decrements in sensory function, threshold testing was performed; probable AD patients had significantly higher olfactory thresholds than controls. To determine if oxidative stress contributes to decreased olfactory function in AD, we localized 3-nitrotyrosine (3-NT) immunoreactivity in OE obtained postmortem from patients with neuropathologically confirmed AD and age-matched controls with brains free of significant neurodegenerative pathology. In AD patients, immunoreactivity was localized in olfactory receptor neurons (ORNs), including dendritic knobs where ion channels that participate in sensory transduction are located, suggesting a direct mechanism for olfactory impairment. In controls, immunoreactivity occurred in blood vessel endothelium, suggesting age-related vascular dysfunction. Immunohistochemistry for CD68, a macrophage scavenger receptor, demonstrated activated macrophages, a source of free radicals contributing to 3-NT formation, in the OE of AD patients but not controls. These results demonstrate increased oxidative stress and modification of ORN proteins that may contribute directly to olfactory impairment in AD patients.     

Cyclic AMP and enzyme secretion by the isolated rabbit pancreas

Summary The isolated rabbit pancreas secreted protein and -amylase at a relatively constant rate during an eight-hour period. Both pancreozymin and acetylcholine caused a massive release of protein and -amylase into pancreatic fluid. Theophylline (10^–2 M) stimulated protein secretion in vitro maximally by 155%, while for -amylase secretion a maximal average stimulation of 80% was observed. Theobromine (10^–2 M) exerted a slight increase on enzyme secretion, equal to that given by 10^–3 M theophylline. Cyclic AMP (10^–3M) also increased enzyme secretion. For protein secretion a maximal average stimulation of 64% was observed, while -amylase secretion was maximally stimulated by 31%. Theophylline (10^–2 M) potentiated the stimulatory effect of pancreozymin (8.5 U/l) on pancreatic enzyme secretion. These results indicate that cyclic AMP is a mediator in enzyme secretion.     

Cyclic AMP mediates serotonin-induced synaptic enhancement of lateral giant interneuron of the crayfish

The lateral giant (LG)-mediated escape behavior of the crayfish habituates readily on repetitive sensory stimulation. Recent studies suggested that the biogenic amines serotonin and octopamine modulate the time course of recovery and/or re-depression of the LG response after habituation. However, little is known of how serotonin and octopamine effect LG habituation and what second-messenger cascades they may activate. To investigate the effect of biogenic amines on LG habituation, serotonin and octopamine were superfused before presenting repetitive sensory stimulation. Serotonin and octopamine increased the number of stimuli needed to habituate the LG response. Their effects were mimicked by mixed application of a cAMP analogue [8-(4-chlorophenylthio)-cAMP (CPT-cAMP)] and a phosphodiesterase inhibitor [3-isobutyl-1-methylxanthine (IBMX)] but not by a cGMP analogue (8-bromoguanosine 3',5'-cyclic monophosphate). Perfusion of the adenylate cyclase inhibitor (SQ22536) abolished the effect of serotonin but not that of octopamine. To investigate the site of action of each biogenic amines in the neural circuit meditating LG escape, the effect of drugs on directly and indirectly elicited postsynaptic potentials in LG was investigated. Serotonin, octopamine, and a mixture of CPT-cAMP and IBMX increased both the direct and indirect synaptic inputs. Simultaneous application of SQ22536 abolished the effect of serotonin on both inputs but did not block the effect of octopamine. Direct injection of the cAMP analogue (Sp-isomer of adenosine-3',5'-cyclic monophosphorothioate) into LG increased both the direct and indirect inputs to LG. These results indicate that serotonin mediates an increase in cAMP levels in LG, but octopamine acts independently of cAMP and cGMP.     

Functional properties of vertebrate olfactory receptor neurons

The interaction of an odorant with the chemosensitive membrane of olfactory receptor neurons initiates a sequence of molecular and membrane events leading to sensory transduction, impulse initiation, and the transmission of sensory information to the brain. The main steps in this sequence are summarized in Figure 6. Several lines of evidence support the hypothesis that the initial molecular events and subsequent stages of transduction are mediated by odorant receptor sites and associated ion channels located in the membrane of the cilia and apical dendritic knob of the olfactory receptor neuron. Similarly, the membrane events associated with impulse initiation and propagation are mediated by voltage-gated channels located in the initial axonal segment and the axolemma. The ionic and electrical events associated with the proposed sequence have been characterized in general using a variety of experimental techniques. The identification, localization, and sequence of membrane events are consistent with the neurophysiological properties observed in specific regions of the bipolar receptor neuron. The influence of other cells in the primary olfactory pathway such as the sustentacular cells in the olfactory epithelium, the Schwann cells in the olfactory nerve, and the astrocytes in the olfactory nerve layer in the olfactory bulb on the physiological activity of the olfactory receptor neuron is an emerging area of research interests. The general principles derived from the experimental results described in this review provide only a framework that is both incomplete and of necessity somewhat speculative. As noted in the Introduction, the multidisciplinary study of the primary olfactory pathway is undergoing a renaissance of research interest. The application of modern biophysical, cell, and molecular biological techniques to the basic issues of odorant recognition and membrane excitability will clarify the speculations and lead to the establishment of new hypotheses. Three broad areas of research will benefit from such studies. First, the application of biophysical techniques will lead to a detailed characterization of the membrane properties and associated ion conductance mechanisms. Second, the isolation and biochemical characterization of intrinsic membrane and cytosolic proteins associated with odorant recognition, sensory transduction, and the subsequent electrical events will result from the utilization of cell and molecular biological techniques.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

Patterned response to odor in mammalian olfactory bulb: the influence of intensity

Responses of single neurons in the olfactory bulb of anesthetized hamsters were recorded extracellularly while odors of defined concentration and time course were delivered to the olfactory system at constant flow. Responses could be either excitatory or suppressive, as judged by the first distinguishable change in firing rate during odor delivery. However, when the time course of the response was examined in more detail, approximately one-third of all tests and one-half of the tests at high concentration resulted in complex temporal patterns of firing rate that involved both increases and decreases with respect to spontaneous activity. Approximately two-thirds of all tests produced responses where increased firing rate preceded any distinguishable suppression. Excitatory and suppressive responses were each classified into four groups according to their temporal patterns. Different patterns were not equally represented in the data and the proportions of patterns elicited by the same odor changed with stimulus intensity. Complex responses, where temporal patterns included periods of firing rate above and below spontaneous rate, were increasingly common and intensity was increased. Magnitude of response is difficult to define when a single response includes both increases and decreases of firing rate but more than half of the neurons that responded to more than one stimulus concentration clearly had nonmonotonic intensity-response functions. Forty-one out of 101 neurons were classified as output cells because they could be driven at short constant latency by lateral olfactory tract stimulation. Their responses were not clearly different from the remaining cells that could not be classified as output cells. The contribution of the inhibitory circuits of the olfactory bulb to the generation of patterned response and to changes in pattern with intensity are discussed. The lateral inhibitory circuits of the bulb appear to be sufficient to explain the data presented here.     

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.     

Neonatal handling and the maternal odor preference in rat pups: Involvement of monoamines and cyclic AMP response element-binding protein pathway in the olfactory bulb

Early-life environmental events, such as the handling procedure, can induce long-lasting alterations upon several behavioral and neuroendocrine systems. However, the changes within the pups that could be causally related to the effects in adulthood are still poorly understood. In the present study, we analyzed the effects of neonatal handling on behavioral (maternal odor preference) and biochemical (cyclic AMP response element-binding protein (CREB) phosphorylation, noradrenaline (NA), and serotonin (5-HT) levels in the olfactory bulb (OB)) parameters in 7-day-old male and female rat pups. Repeated handling (RH) abolished preference for the maternal odor in female pups compared with nonhandled (NH) and the single-handled (SH) ones, while in RH males the preference was not different than NH and SH groups. In both male and female pups, RH decreased NA activity in the OB, but 5-HT activity increased only in males. Since preference for the maternal odor involves the synergic action of NA and 5-HT in the OB, the maintenance of the behavior in RH males could be related to the increased 5-HT activity, in spite of reduction in the NA activity in the OB. RH did not alter CREB phosphorylation in the OB of both male and females compared with NH pups. The repeated handling procedure can affect the behavior of rat pups in response to the maternal odor and biochemical parameters related to the olfactory learning mechanism. Sex differences were already detected in 7-day-old pups. Although the responsiveness of the hypothalamic–pituitary–adrenal axis to stressors is reduced in the neonatal period, environmental interventions may impact behavioral and biochemical mechanisms relevant to the animal at that early age.