Cyclic nucleotide-gated ion channels in olfactory sensory neurons (OSNs) are hypothesized to play a critical role in olfaction. However, it has not been demonstrated that the cAMP signaling is required for olfactory-based behavioral responses, and the contributions of specific adenylyl cyclases to olfaction have not been defined. Here, we report the presence of adenylyl cyclases 2, 3, and 4 in olfactory cilia. To evaluate the role of AC3 in olfactory responses, we disrupted the gene for AC3 in mice. Interestingly, electroolfactogram (EOG) responses stimulated by either cAMP- or inositol 1,4,5-triphosphate- (IP3-) inducing odorants were completely ablated in AC3 mutants, despite the presence of AC2 and AC4 in olfactory cilia. Furthermore, AC3 mutants failed several olfaction-based behavioral tests, indicating that AC3 and cAMP signaling are critical for olfactory-dependent behavior.

The sense of smell is highly evolved in mammals, allowing discrimination between a vast number of odorants, with detection thresholds as low as 10(-17) M (ref. 1). Although several features of mammalian olfactory transduction have been revealed by biochemical and molecular biological studies, the odorant-induced membrane current has remained elusive. In amphibians this current is mediated by cyclic-nucleotide-gated channels, which depolarize the cell by Na+ and Ca+ influx and consequent Cl- efflux through Ca(2+)-dependent Cl- channels. The Cl- current may be absent in mammals, however, because its proposed role is linked to the aquatic habitat of amphibians. Here we show that the transduction current in rat olfactory receptor cells is initiated by cyclic-nucleotide-gated channels. The Cl- current is also present and endows the transduction current with a steep sigmoidal dependence on cyclic AMP concentration in both rat and in an amphibian, indicating a new function for the Cl- channel: nonlinear amplification of the transduction signal, whereby suprathreshold responses are boosted relative to basal transduction noise.

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Olfactory neurons expressing the same odorant receptor converge to a small number of glomeruli in the olfactory bulb. In turn, mitral and tufted cells receive and relay this information to higher cortical regions. In other sensory systems, correlated neuronal activity is thought to refine synaptic connections during development. We asked whether the pattern of connections between olfactory sensory axons and mitral cell dendrites is affected when odor-evoked signaling is eliminated in mice lacking functional olfactory cyclic nucleotide-gated (CNG) channels. We demonstrate that olfactory sensory axons converge normally in the CNG channel mutant background. We further show that the pruning of mitral cell dendrites, although slowed during development, is ultimately unperturbed in mutant animals. Thus, the olfactory CNG channel-and by inference correlated neural activity--is not required for generating synaptic specificity in the olfactory bulb.

The mammalian olfactory bulb is a geometrically organized signal-processing array that utilizes lateral inhibitory circuits to transform spatially patterned inputs. A major part of the lateral circuitry consists of extensively radiating secondary dendrites of mitral cells. These dendrites are bidirectional cables: they convey granule cell inhibitory input to the mitral soma, and they conduct backpropagating action potentials that trigger glutamate release at dendrodendritic synapses. This study examined how mitral cell firing is affected by inhibitory inputs at different distances along the secondary dendrite and what happens to backpropagating action potentials when they encounter inhibition. These are key questions for understanding the range and spatial dependence of lateral signaling between mitral cells. Backpropagating action potentials were monitored in vitro by simultaneous somatic and dendritic whole cell recording from individual mitral cells in rat olfactory bulb slices, and inhibition was applied focally to dendrites by laser flash photolysis of caged GABA (2.5-microm spot). Photolysis was calibrated to activate conductances similar in magnitude to GABA(A)-mediated inhibition from granule cell spines. Under somatic voltage-clamp with CsCl dialysis, uncaging GABA onto the soma, axon initial segment, primary and secondary dendrites evoked bicuculline-sensitive currents (up to -1.4 nA at -60 mV; reversal at approximatety 0 mV). The currents exhibited a patchy distribution along the axon and dendrites. In current-clamp recordings, repetitive firing driven by somatic current injection was blocked by uncaging GABA on the secondary dendrite approximately 140 microm from the soma, and the blocking distance decreased with increasing current. In the secondary dendrites, backpropagated action potentials were measured 93-152 microm from the soma, where they were attenuated by a factor of 0.75 +/- 0.07 (mean +/- SD) and slightly broadened (1.19 +/- 0.10), independent of activity (35-107 Hz). Uncaging GABA on the distal dendrite had little effect on somatic spikes but attenuated backpropagating action potentials by a factor of 0.68 +/- 0.15 (0.45-0.60 microJ flash with 1-mM caged GABA); attenuation was localized to a zone of width 16.3 +/- 4.2 microm around the point of GABA release. These results reveal the contrasting actions of inhibition at different locations along the dendrite: proximal inhibition blocks firing by shunting somatic current, whereas distal inhibition can impose spatial patterns of dendrodendritic transmission by locally attenuating backpropagating action potentials. The secondary dendrites are designed with a high safety factor for backpropagation, to facilitate reliable transmission of the outgoing spike-coded data stream, in parallel with the integration of inhibitory inputs.

We report the visualization of NO production using fluorescence in tissue slices of the mouse main olfactory bulb. This discovery was possible through the use of a novel, cell-trappable probe for intracellular nitric oxide detection based on a symmetric scaffold with two NO-reactive sites. Ester moieties installed onto the fluorescent probe are cleaved by intracellular esterases to yield the corresponding negatively charged, cell-impermeable acids. The trappable probe Cu 2 ðFL2EÞ and the membrane-impermeable acid derivative Cu 2 ðFL2AÞ respond rapidly and selectively to NO in buffers that simulate biological conditions, and application of Cu 2 ðFL2EÞ leads to detection of endogenously produced NO in cell cultures and olfactory bulb brain slices.fluorescent sensing | NO | olfaction | trappable probe | fluorescence microscopy N itric oxide (NO) is important for biological signaling. It activates soluble guanylyl cyclase, initiating a signaling cascade that promotes vascular smooth muscle dilation (1-3). Nitric oxide produced in the nervous system has been implicated in neurotransmission (4), and the immune system generates NO as a defense against pathogens (5). Unregulated nitric oxide production has been associated with pathological conditions such as cancer, ischemia, septic shock, inflammation, and neurodegeneration (6).Because of its various biological consequences, investigating the generation, translocation, and utilization of NO continues to be an active area of research. A major limitation to advances in the field, however, has been the dearth of selective tools for visualization of biological NO, for example by fluorescence microscopy. Detection of nitric oxide offers many challenges. NO reacts rapidly in vivo with dioxygen, oxygen-generated radicals such as superoxide, amines, thiols, and metal centers (7,8). It also diffuses readily from its point of origin (9), making rapid detection desirable for uncovering both its production site and function. Moreover, NO is produced at concentrations as low as ∼100 picomolar, so it is essential to have an NO probe with a low detection limit (10). Fluorescent sensors can be designed to accommodate the properties of NO under physiological conditions, making this technique particularly valuable for in vivo nitric oxide imaging.Transition metal complexes have been investigated as platforms for NO detection (11). The strategy is to incorporate a fluorophore into a ligand that is quenched either by intracellular photoinduced electron transfer and/or by coordination to a paramagnetic or heavy metal ion. Fluorescence is restored by reduction of the metal and/or displacement of the ligand upon reaction of the probe with NO. CuFL1 (Fig. 1) is an excellent example of such a metal-based cellular NO imaging agent (12, 13). CuFL1 satisfies many of the requirements of a good sensor. It is nontoxic, cell membrane permeable, has low energy excitation and emission wavelengths, responds directly and selectively to NO, and exhibits dramatic fluorescence enhancement upon reaction with...

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An odor-stimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] is thought to mediate olfactory transduction in vertebrates. However, it is not known whether the adenylate cyclase serves this function for all odorants or for only certain classes of odorants. To investigate this question, we have compared the abilities of 35 odorants to stimulate the adenylate cyclase and to elicit an electrophysiological response. We report a strong positive correlation between the magnitude of adenylate cyclase stimulation and the summated electrical response of the olfactory epithelium (electro-olfactogram) evoked by individual odorants. We also show that the adenylate cyclase stimulator forskolin equally attenuates the electro-olfactogram response for all odorants tested. These data provide evidence that the adenylate cyclase mediates transduction for a wide variety of odorants.Olfactory receptor cells respond to odors by an increase in membrane conductance, leading to membrane depolarization and the generation of action potentials (reviewed in refs. 1 and 2). The mechanism(s) by which odors regulate the membrane conductance is not known with certainty; however, a considerable body of evidence indicates that cyclic AMP serves as an intracellular messenger in this process. For example, olfactory cilia contain an odor-stimulated adenylate cyclase (3)(4)(5). In addition, we have described a cyclic nucleotide-gated conductance that could translate the odorinduced rise in cyclic AMP concentration into a conductance increase (6). The involvement of the cyclic AMP pathway is further suggested by the effects of pharmacological agents on the electrical responses to odors, measured with transepithelial voltage and current-recordings (7)(8)(9).A key question concerning the cyclic AMP mechanism is whether it mediates transduction for all odorants or only certain classes of odorants. We (Union Beach, NJ). Forskolin and 1,9-dideoxyforskolin were purchased from Calbiochem.The EOG was measured from excised bullfrog olfactory epithelia, which were mounted in a perfusion chamber. Frogs were decapitated and pithed, and the dorsal surface of the nasal cavity was soaked in Ringer's solution for ca. 2 hr at room temperature. The 2-hr soak in Ringer's solution was found to give EOG responses of larger amplitudes. The olfactory epithelium was then dissected away from the remaining portion of the skull and mounted, apical (ciliated) surface up, in a Plexiglas recording chamber (15), and the edges were sealed with vacuum grease. The apical surface was continuously superfused with Ringer's solution via a plug, which sealed the chamber from above. The plug had a central port for delivery of fresh Ringer's solution (flow rate 2.0 ml/min) and a concentric array of peripheral exit ports to maintain an approximately radial flow pattern. The exposed area of the epithelium was 12 mm2, which utilized most ofthe dissected tissue. The Ringer's solution contained 100 mM NaCl, 2.5 mM KCl, 10 mM NaHCO3, 1 mM CaCl2, 1 mM MgSO4, an...