Parallel processing of social signals by the mammalian main and accessory olfactory systems.

The mammalian olfactory system has evolved complex mechanisms to detect a vast range of molecular cues. In rodents, the olfactory system comprises several distinct subsystems. Current interest has focused on the exact role that each of these subsystems plays in detecting molecular information and regulating chemosensory-dependent behaviors. Here, we summarize recent results showing that the mouse main and accessory olfactory systems detect, at least in part, overlapping sets of social chemosignals. These findings give rise to a model that involves parallel processing of the same molecular cues in both systems. Together with previous work, this model will lead to a better understanding of the general organization of chemical communication in mammals and give a new direction for future experiments.

Central role of the CNGA4 channel subunit in Ca2+-calmodulin-dependent odor adaptation.

Heteromultimeric cyclic nucleotide-gated (CNG) channels play a central role in the transduction of odorant signals and subsequent adaptation. The contributions of individual subunits to native channel function in olfactory receptor neurons remain unclear. Here, we show that the targeted deletion of the mouse CNGA4 gene, which encodes a modulatory CNG subunit, results in a defect in odorant-dependent adaptation. Channels in excised membrane patches from the CNGA4 null mouse exhibited slower Ca2+-calmodulin-mediated channel desensitization. Thus, the CNGA4 subunit accelerates the Ca2+-mediated negative feedback in olfactory signaling and allows rapid adaptation in this sensory system.

The cellular and molecular basis of odor adaptation.

An important recent advance in the understanding of odor adaptation has come from the discovery that complex mechanisms of odor adaptation already take place at the earliest stage of the olfactory system, in the olfactory cilia. At least two rapid forms and one persistent form of odor adaptation coexist in vertebrate olfactory receptor neurons. These three different adaptation phenomena can be dissected on the basis of their different onset and recovery time courses and their pharmacological properties, indicating that they are controlled, at least in part, by separate molecular mechanisms. Evidence is provided for the involvement of distinct molecular steps in these forms of odor adaptation, including Ca(2+) entry through cyclic nucleotide-gated (CNG) channels, Ca(2+)-dependent CNG channel modulation, Ca(2+)/calmodulin kinase II-dependent attenuation of adenylyl cyclase, and the activity of the carbon monoxide/cyclic GMP second messenger system. Identification of these molecular steps may help to elucidate how the olfactory system extracts temporal and intensity information and to which extent odor perception is influenced by the different mechanisms underlying adaptation.

Blocking adenylyl cyclase inhibits olfactory generator currents induced by "IP(3)-odors".

Vertebrate olfactory receptor neurons (ORNs) transduce odor stimuli into electrical signals by means of an adenylyl cyclase/cAMP second messenger cascade, but it remains widely debated whether this cAMP cascade mediates transduction for all odorants or only certain odor classes. To address this problem, we have analyzed the generator currents induced by odors that failed to produce cAMP in previous biochemical assays but instead produced IP(3) ("IP(3)-odors"). We show that in single salamander ORNs, sensory responses to "cAMP-odors" and IP(3)-odors are not mutually exclusive but coexist in the same cells. The currents induced by IP(3)-odors exhibit identical biophysical properties as those induced by cAMP odors or direct activation of the cAMP cascade. By disrupting adenylyl cyclase to block cAMP formation using two potent antagonists of adenylyl cyclase, SQ22536 and MDL12330A, we show that this molecular step is necessary for the transduction of both odor classes. To assess whether these results are also applicable to mammals, we examine the electrophysiological responses to IP(3)-odors in intact mouse main olfactory epithelium (MOE) by recording field potentials. The results show that inhibition of adenylyl cyclase prevents EOG responses to both odor classes in mouse MOE, even when "hot spots" with heightened sensitivity to IP(3)-odors are examined.

Ultrasensitive pheromone detection by mammalian vomeronasal neurons.

The vomeronasal organ (VNO) is a chemoreceptive organ that is thought to transduce pheromones into electrical responses that regulate sexual, hormonal and reproductive function in mammals. The characteristics of pheromone signal detection by vomeronasal neurons remain unclear. Here we use a mouse VNO slice preparation to show that six putative pheromones evoke excitatory responses in single vomeronasal neurons, leading to action potential generation and elevated calcium entry. The detection threshold for some of these chemicals is remarkably low, near 10(-11) M, placing these neurons among the most sensitive chemodetectors in mammals. Using confocal calcium imaging, we map the epithelial representation of the pheromones to show that each of the ligands activates a unique, nonoverlapping subset of vomeronasal neurons located in apical zones of the epithelium. These neurons show highly selective tuning properties and their tuning curves do not broaden with increasing concentrations of ligand, unlike those of receptor neurons in the main olfactory epithelium. These findings provide a basis for understanding chemical signals that regulate mammalian communication and sexual behaviour.

Cyclic GMP evoked calcium transients in olfactory receptor cell growth cones.

Nitric oxide-induced calcium transients in growth cones are believed to be mediated by cyclic nucleotides. Because nitric oxide is thought to influence the development of olfactory receptor cells (ORCs), we have begun to explore the effect of cyclic nucleotides on ORC growth cones. Cultured ORCs were loaded with fluo-3 AM and confocal imaging was employed to monitor calcium transients following cyclic nucleotide-gated channel activation. Application of 8-bromo-cGMP at the growth cone caused transient increases in fluorescence which were restricted to the growth cone and lasted tens of seconds. The signal was abolished by LY83583, an inhibitor of cyclic nucleotide-gated channels. 8-Bromo-cGMP also inhibited further extension of growth cones. The data indicate that ORC growth cones exhibit cGMP-dependent calcium transients that are consistent with those generated by cyclic nucleotide-gated channels.

Amplification of odor-induced Ca(2+) transients by store-operated Ca(2+) release and its role in olfactory signal transduction.

A critical role of Ca(2+) in vertebrate olfactory receptor neurons (ORNs) is to couple odor-induced excitation to intracellular feedback pathways that are responsible for the regulation of the sensitivity of the sense of smell, but the role of intracellular Ca(2+) stores in this process remains unclear. Using confocal Ca(2+) imaging and perforated patch recording, we show that salamander ORNs contain a releasable pool of Ca(2+) that can be discharged at rest by the SERCA inhibitor thapsigargin and the ryanodine receptor agonist caffeine. The Ca(2+) stores are spatially restricted; emptying produces compartmentalized Ca(2+) release and capacitative-like Ca(2+) entry in the dendrite and soma but not in the cilia, the site of odor transduction. We deplete the stores to show that odor stimulation causes store-dependent Ca(2+) mobilization. This odor-induced Ca(2+) release does not seem to be necessary for generation of an immediate electrophysiological response, nor does it contribute significantly to the Ca(2+) transients in the olfactory cilia. Rather, it is important for amplifying the magnitude and duration of Ca(2+) transients in the dendrite and soma and is thus necessary for the spread of an odor-induced Ca(2+) wave from the cilia to the soma. We show that this amplification process depends on Ca(2+)-induced Ca(2+) release. The results indicate that stimulation of ORNs with odorants can produce Ca(2+) mobilization from intracellular stores without an immediate effect on the receptor potential. Odor-induced, store-dependent Ca(2+) mobilization may be part of a feedback pathway by which information is transferred from the distal dendrite of an ORN to its soma.

Impaired odor adaptation in olfactory receptor neurons after inhibition of Ca2+/calmodulin kinase II.

Odor adaptation in vertebrate olfactory receptor neurons (ORNs) is commonly attributed to feedback modulation caused by Ca(2+) entry through the transduction channels, but it remains unclear and controversial whether this Ca(2+)-mediated adaptation resides in the cAMP-gated channel alone or whether other molecules of the transduction cascade are modulated as well. Attenuation of adenylyl cyclase activity by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) has also been proposed as a mechanism for adaptation. To test this in intact ORNs, we have compared the properties of adaptation induced by a sustained (8 sec) or brief (100 msec) odor stimulus. Although adaptation induced by both types of stimuli occurs downstream from the odor receptors and is Ca(2+)-dependent, only adaptation induced by a sustained pulse involves alterations in the odor response kinetics, consistent with a reduction in the rate of adenylyl cyclase activation. By disrupting CaMKII to block adenylyl cyclase attenuation using a specific peptide inhibitor of CaMKII, autocamtide-2-related inhibitory peptide (AIP), we show that this reaction is necessary for odor adaptation in vivo. With CaMKII disrupted, adaptation induced by a sustained stimulus is significantly impaired: the onset rate of adaptation is decreased by threefold, and the recovery rate from adaptation is increased by up to sixfold. In contrast, adaptation induced by a brief odor pulse is unaffected, demonstrating that the effect of AIP must be highly specific. The results indicate that CaMKII controls the temporal response properties of ORNs during odor adaptation. We propose that CaMKII plays a prominent role in odor perception.

Imaging odor-induced calcium transients in single olfactory cilia: specificity of activation and role in transduction.

The possibility that odor stimuli trigger distinct Ca2+ elevations within the cilia of vertebrate olfactory receptor neurons (ORNs) is a widely proposed concept. However, because of the small size of the olfactory cilia, the existence and properties of such Ca2+ elevations and their role in odor transduction are still unknown. We investigate odor-induced Ca2+ changes in individual olfactory cilia from salamander using the Ca2+ indicator dye fluo-3 in combination with laser scanning confocal microscopy. Single brief applications of odor ligand produce highly localized Ca2+ elevations in individual cilia lasting for several seconds. These Ca2+ signals originate in the cilia and depend entirely on Ca2+ entry through ciliary cyclic nucleotide-gated ion channels. The odor specificity of the Ca2+ rises implies a receptor-operated mechanism underlying odor detection. Each of the cilia on a receptor neuron functions as an independent biochemical compartment that can detect odorants and produce a Ca2+ transient with remarkably uniform properties in terms of kinetics and odor specificity. The rate of recovery of the odor-induced Ca2+ transients matches recovery from a short-term form of odor adaptation. Application of the membrane-permeant intracellular Ca2+ chelator BAPTA AM eliminates this odor adaptation. The results indicate that an olfactory cilium serves as a basic functional unit at the input level of the olfactory system, controlling both the specificity and sensitivity of odor detection.

Role of cyclic GMP in olfactory transduction and adaptation.

The detection of odor molecules by vertebrate olfactory receptor neurons (ORNs) involves signal transduction mechanisms that are thought to occur primarily through a cyclic adenosine monophosphate (cAMP)-mediated second messenger pathway. There has been intense debate whether cAMP is the sole second messenger responsible for all excitation and adaptation. The recent identification of a distinct form of odor adaptation that depends on the carbon monoxide/cyclic guanosine monophosphate (cGMP) second messenger system demonstrates that cAMP alone cannot account for all phases of adaptation and that multiple second messenger pathways exist in ORNs to perform distinct but closely related olfactory functions.

Visualizing odor detection in olfactory cilia by calcium imaging.

To visualize odor detection in individual cilia of olfactory sensory neurons we have developed a new approach by using high-resolution calcium imaging techniques. Laser scanning confocal microscopy revealed, for the first time, that odor stimuli induce transient Ca2+ elevations in single olfactory cilia. Pharmacological analysis indicates that these Ca2+ signals depend entirely on Ca2+ entry through activated cyclic nucleotide-gated (CNG) channels. This novel approach enables us to monitor the initial steps leading to olfactory perception in a spatially and temporally resolved manner.

Calcium entry through cyclic nucleotide-gated channels in individual cilia of olfactory receptor cells: spatiotemporal dynamics.

Transient elevations of intracellular Ca2+ play an important role in regulating the sensitivity of olfactory transduction, but such elevations have not been demonstrated in the olfactory cilia, which are the site of primary odor transduction. To begin to understand Ca2+ signaling in olfactory cilia, we used high-resolution imaging techniques to study the Ca2+ transients that occur in salamander olfactory receptor neurons (ORNs) as a result of cyclic nucleotide-gated (CNG) channel activation. To visualize ciliary Ca2+ signals, we loaded ORNs with the Ca2+ indicator dye Fluo-3 AM and measured fluorescence with a laser scanning confocal microscope. Application of the phosphodiesterase inhibitor IBMX increased fluorescence in the cilia and other neuronal compartments; the ciliary signal occurred first and was more transient. This signal could be abolished by lowering external Ca2+ or by applying LY83583, a potent blocker of CNG channels, indicating that Ca2+ entry through CNG channels was the primary source of fluorescence increases. Direct activation of CNG channels with low levels of 8-Br-cGMP (1 microM) led to tonic Ca2+ signals that were restricted locally to the cilia and the dendritic knob. Elevated external K+, which depolarizes cell membranes, increased fluorescence signals in the cell body and dendrite but failed to increase ciliary Ca2+ fluorescence. The results demonstrate the existence and spatiotemporal properties of Ca2+ transients in individual olfactory cilia and implicate CNG channels as a major pathway for Ca2+ entry into ORN cilia during odor transduction.

Identification of a long-lasting form of odor adaptation that depends on the carbon Monoxide/cGMP second-messenger system.

The diffusible messenger carbon monoxide (CO) has been proposed to mediate endogenous cyclic guanosine 3',5'-monophosphate (cGMP) formation and sensory adaptation in vertebrate olfactory receptor neurons (ORNs). We have identified and characterized a long-lasting form of odor response adaptation (LLA) that operates at the level of isolated salamander ORNs and does not require any interactions from other cells. Manifestations of LLA are seen in reduced amplitude and prolonged kinetics of the cAMP-mediated excitatory odor response and the generation of a persistent current component that lasts for several minutes and is attributable to cyclic nucleotide-gated (CNG) channel activation by cGMP. Because these effects can be mimicked by micromolar amounts of exogenous cGMP or CO, we applied various inhibitors of cGMP formation. LLA is abolished selectively by heme oxygenase inhibitors known to prevent CO release and cGMP formation in ORNs, whereas odor excitation remains unaffected. In contrast, blockers of nitric oxide synthase are unable to eliminate LLA. Several controls rule out a contribution of nonspecific actions to the effects of CO inhibitors. The results indicate that endogenous CO/cGMP signals contribute to olfactory adaptation and underlie the control of gain and sensitivity of odor transduction. The findings offer a mechanism by which a single, brief odor stimulus can be translated into long-lasting intracellular changes that could play an important role in the perceptual adaptation to odors, and explain the longstanding puzzle that the olfactory CNG channels can be gated by both cAMP and cGMP.

Modulation by cyclic GMP of the odour sensitivity of vertebrate olfactory receptor cells.

Recent evidence has indicated a significant role for the cGMP second messenger system in vertebrate olfactory transduction but no clear functions have been identified for cGMP so far. Here, we have examined the effects of 8-Br-cGMP and carbon monoxide (CO) on odour responses of salamander olfactory receptor neurons using perforated patch recordings. We report that 8-Br-cGMP strongly down-regulates the odour sensitivity of the cells, with a K1/2 of 460 nM. This adaptation-like effect can be mimicked by CO, an activator of soluble guanylyl cyclase, with a K1/2 of 1 microM. Sensitivity modulation is achieved through a regulatory chain of events in which cGMP stimulates a persistent background current due to the activation of cyclic nucleotide-gated channels. This in turn leads to sustained Ca2+ entry providing a negative feedback signal. One consequence of the Ca2+ entry is a shift to the right of the stimulus-response curve and a reduction in saturating odour currents. Together, these two effects can reduce the sensory generator current by up to twenty-fold. Thus, cGMP functions to control the gain of the G-protein coupled cAMP pathway. Another consequence of the action of cGMP is a marked prolongation of the odour response kinetics. The effects of CO/cGMP are long-lasting and can continue for minutes. Hence, we propose that cGMP helps to prevent saturation of the cell's response by adjusting the operational range of the cAMP cascade and contributes to olfactory adaptation by decreasing the sensitivity of olfactory receptor cells to repeated odour stimuli.

Block of cyclic nucleotide-gated channels in salamander olfactory receptor neurons by the guanylyl cyclase inhibitor LY83583.

1. Using whole cell voltage-clamp recordings, the guanylyl cyclase inhibitor LY83583 [6-(phenylamino)-5,8-quinolinedione] is shown to act as a potent blocker of cyclic nucleotide-gated (CNG) channels in isolated olfactory receptor neurons (ORNs) of the tiger salamander. 2. Under our experimental conditions, onset of the blockade by LY83583 occurs on the time scale of seconds and is completely reversed upon wash-out of the drug. Dose-response curves reveal a Kd of 1.4 microM (at -60 mV). Other data suggest that LY83583 acts within the CNG channel pore and that the channels must be in an activated state before the drug can exert its effect. 3. It appears that LY83583 can act on both CNG channels and soluble guanylyl cyclase (sGC) and that these two effects can be distinguished by their different recovery behaviors. The LY83583-induced blockade of CNG channels activated directly by guanosine 3',5' cyclic monophosphate (cGMP) is rapidly reversible (with a recovery time constant of approximately 3 s), whereas previous results have shown that no recovery is obtained during minute-long washing periods when the channels are activated indirectly through exogenous carbon monoxide application, which acts as a stimulator of sGC in ORNs. 4. LY83583 appears to be a novel and useful agent in examining neural functions due to CNG channel responses.

Regulation of cyclic nucleotide-gated channels and membrane excitability in olfactory receptor cells by carbon monoxide.

1. The effect of the putative neural messenger carbon monoxide (CO) and the role of the cGMP second-messenger system for olfactory signal generation was examined in isolated olfactory receptor neurons (ORNs) of the tiger salamander. 2. With the use of whole cell voltage-clamp recordings in combination with a series of ionic and pharmological tests, it is demonstrated that exogenously applied CO is a potent activator (K1/2 = 2.9 microM) of cyclic nucleotide-gated (CNG) channels previously described to mediate odor transduction. 3. Several lines of evidence suggest that CO mediates its effect through stimulation of a soluble guanylyl cyclase (sGC) leading to formation of the second-messenger cGMP. This conclusion is based on the findings that CO responses show an absolute requirement for guanosine 5'-triphosphate (GTP) in the internal solution, that no direct effect of CO on CNG currents in the absence of GTP is detectable, and that a blocker of sGC activation, LY85383 (10 microM), completely inhibits the CO response. 4. The dose-response curve for cGMP at CNG channels is used as a calibration to provide a quantitative estimate of the CO-stimulated cGMP formation. This analysis implies that CO is a potent activator of olfactory sGC. 5. Perforated patch recordings using amphotericin B demonstrate that low micromolar doses of CO effectively depolarize the membrane potential of ORNs through tonic activation of CNG channels. This effect in turn regulates excitable and adaptive properties of ORNs and modulates neuronal responsiveness. 6. These data argue for an important role of the cGMP pathway in olfactory signaling and support the idea that CO may function as a diffusible messenger in the olfactory system.

A calcium-permeable cGMP-activated cation conductance in hippocampal neurons.

Whole-cell patch clamp recordings detected a previously unidentified cGMP-activated membrane conductance in cultured rat hippocampal neurons. This conductance is nonselectively permeable for cations and is completely but reversibly blocked by external Cd2+. The Ca2+ permeability of the hippocampal cGMP-activated conductance was examined in detail, indicating that the underlying ion channels display a high relative permeability for Ca2+. The results indicate that hippocampal neurons contain a cGMP-activated membrane conductance that has some properties similar to the cyclic nucleotide-gated channels previously shown in sensory receptor cells and retinal neurons. In hippocampal neurons this conductance similarly could mediate membrane depolarization and Ca2+ fluxes in response to intracellular cGMP elevation.

Differential effects of heavy metal ions on Ca(2+)-dependent K+ channels.

1. The ability of various divalent metal ions to substitute for Ca2+ in activating distinct types of Ca(2+)-dependent K+ [K+(Ca2+)] channels has been investigated in excised, inside-out membrane patches of human erthrocytes and of clonal N1E-115 mouse neuroblastoma cells using the patch clamp technique. The effects of the various metal ions have been compared and related to the effects of Ca2+. 2. At concentrations between 1 and 100 microM Pb2+, Cd2+ and Co2+ activate intermediate conductance K+(Ca2+) channels in erythrocytes and large conductance K+(Ca2+) channels in neuroblastoma cells. Pb2+ and Co2+, but not Cd2+, activate small conductance K+(Ca2+) channels in neuroblastoma cells. Mg2+ and Fe2+ do not activate any of the K+(Ca2+) channels. 3. Rank orders of the potencies for K+(Ca2+) activation are Pb2+, Cd2+ > Ca2+, Co2+ >> Mg2+, Fe2+ for the intermediate erythrocyte K+(Ca2+) channel, and Pb2+, Cd2+ > Ca2+ > Co2+ >> Mg2+, Fe2+ for the small, and Pb2+ > Ca2+ > Co2+ >> Cd2+, Mg2+, Fe2+ for the large K+(Ca2+) channel in neuroblastoma cells. 4. At high concentrations Pb2+, Cd2+, and Co2+ block K+(Ca2+) channels in erythrocytes by reducing the opening frequency of the channels and by reducing the single channel amplitude. The potency orders of the two blocking effects are Pb2+ > Cd2+, Co2+ >> Ca2+, and Cd2+ > Pb2+, Co2+ >> Ca2+, respectively, and are distinct from the potency orders for activation. 5. It is concluded that the different subtypes of K+(Ca2+) channels contain distinct regulatory sites involved in metal ion binding and channel opening. The K+(Ca2+) channel in erythrocytes appears to contain additional metal ion interaction sites involved in channel block.

Differential role of two Ca(2+)-permeable non-NMDA glutamate channels in rat retinal ganglion cells: kainate-induced cytoplasmic and nuclear Ca2+ signals.

1. The permeability of non-N-methyl-D-aspartate (non-NMDA) glutamate channels to divalent cations and specifically the entry of Ca2+ and subsequent elevations in cytoplasmic and nuclear Ca2+ signals were investigated in cultured neonatal rat retinal ganglion cells using the whole cell patch-clamp technique and Ca2+ imaging with confocal microscopy. In addition, divalent-permeable non-NMDA receptor channels were studied in retinal slices using a Co2+ staining technique. 2. Using Ca2+ (2.5 mM) as the only permeable cation in the external solution, stimulation with 100 microM kainate produced nondesensitizing, nonselective cation currents with either low or high Ca2+ permeability. Both currents were reversibly blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Neurons with the low divalent-permeable currents (type 1) had reversal potentials of -41.5 +/- 4.4 mV (mean +/- SD), and neurons with the high divalent-permeable currents (type 2) had reversal potentials of -22.6 +/- 5.5 mV. The permeability ratio PCa/PCs was 3.3 for the type 1 currents and 8.5 for the type 2 currents, indicating a 2.5-fold greater permeability to Ca2+ for the type 2 non-NMDA glutamate channels. 3. Both types of non-NMDA glutamate channels showed relatively little selectivity between Ca2+ and Co2+. The type 1 neurons had a slightly higher permeability to Co2+ than to Ca2+, whereas the type 2 neurons were equally permeable to both divalent cations. The type 2 neurons had a much higher permeability for both divalent cations compared with the type 1 neurons. 4. Staining for Co2+ uptake through kainate-stimulated non-NMDA glutamate channels in retinal slices provided additional evidence for the presence of the two ganglion cell populations. Activation of the neurons by kainate in conditions isolating the non-NMDA glutamate channel caused differential uptake of Co2+. In contrast, depolarization in the presence of the non-NMDA antagonist CNQX failed to cause Co2+ influx. 5. Imaging experiments using confocal microscopy showed that kainate stimulation induced an increase in intracellular Ca2+ in both types of retinal ganglion cells, but only the type 2 neurons showed a substantial increase in cytoplasmic and nuclear Ca2+ signals. Kainate-induced Ca2+ signals in the type 2 neurons were almost nine times greater than those of the type 1 neurons. 6. When intracellular Ca2+ stores were depleted by brief treatment with thapsigargin, kainate-induced Ca2+ signals in the type 1 neurons were unchanged. However, in the type 2 neurons kainate no longer induced large Ca2+ signals in the cytoplasm and nucleus, despite normal influx of Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)