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...

Non-technical summary Odorants are transported into the nasal cavity upon air inhalation where they are detected by olfactory receptor neurons (ORNs), which transduce the odorant molecules into action potentials. The rate of stimulation thus depends on the chosen breathing frequency, which in mice ranges from 2 to 10 Hz. This poses the question how ORNs respond to rapidly changing stimulation rates. Individual mouse ORNs respond reliably to repetitive 2 Hz stimulations resembling normal breathing, but actually perform much poorer when the stimulation rate is increased to 5 Hz, which is more akin to sniffing. In this case, rarely more than 50% of the stimulations elicit any response, with an increase in odorant concentration further reducing the response rate, becoming zero at high concentrations. This counterintuitive observation can be understood in the framework of an adaptive filter, which allows the animal to selectively alter its ORN output depending on the chosen breathing rate.Abstract Vertebrate olfactory receptor neurons (ORNs) are stimulated in a rhythmic manner in vivo, driven by delivery of odorants to the nasal cavity carried by the inhaled air, making olfaction a sense where animals can control the frequency of stimulus delivery. How ORNs encode repeated stimulation at resting, low breathing frequencies and at increased sniffing frequencies is not known, nor is it known if the olfactory transduction cascade is accurate and fast enough to follow high frequency stimulation. We investigated mouse olfactory responses to stimulus frequencies mimicking odorant exposure during low (2 Hz) and high (5 Hz) frequency sniffing. ORNs reliably follow low frequency stimulations with high fidelity by generating bursts of action potentials at each stimulation at intermediate odorant concentrations, but fail to do so at high odorant concentrations. Higher stimulus frequencies across all odorant concentrations reduced the likelihood of action potential generation, increased the latency of response, and decreased the reliability of encoding the onset of stimulation. Thus an increase in stimulus frequency degrades and at high odorant concentrations entirely prevents action potential generation in individual ORNs, causing reduced signalling to the olfactory bulb. These results demonstrate that ORNs do not simply relay timing and concentration of an odorous stimulus, but also process and modulate the stimulus in a frequency-dependent manner which is controlled by the chosen sniffing rate.

The mammalian olfactory bulb receives multiple modulatory inputs, including a cholinergic input from the basal forebrain. Understanding the functional roles played by the cholinergic input requires an understanding of the cellular mechanisms it modulates. In an in vitro olfactory bulb slice preparation we demonstrate cholinergic muscarinic modulation of glutamate release onto granule cells that results in gamma-aminobutyric acid (GABA) release onto mitral/tufted cells. We demonstrate that the broad-spectrum cholinergic agonist carbachol triggers glutamate release from mitral/tufted cells that activates both AMPA and NMDA receptors on granule cells. Activation of the granule cell glutamate receptors leads to calcium influx through voltage-gated calcium channels, resulting in spike-independent, asynchronous GABA release at reciprocal dendrodendritic synapses that granule cells form with mitral/tufted cells. This cholinergic modulation of glutamate release persists through much of postnatal bulbar development, suggesting a functional role for cholinergic inputs from the basal forebrain in bulbar processing of olfactory inputs and possibly in postnatal development of the olfactory bulb.

The significance of endoplasmic reticulum (ER) store calcium in modulating transmitter release is slowly gaining recognition. One transmitter system that might play an important role in store calcium modulation of transmitter release in the CNS is acetylcholine (ACh). The main olfactory bulb (OB) receives rich cholinergic innervation from the horizontal limb of the diagonal band of Broca and blocking cholinergic signaling in the bulb inhibits the ability of animals to discriminate between closely related odors. Here we show that exposing OB slices to carbamylcholine (CCh), a hydrolysis-resistant analog of Ach, increases gamma-aminobutyric acid (GABA) release at dendrodendritic synapses onto the mitral cells. This increase in transmitter release is mediated by the activation of the M1 class of muscarinic receptors and requires the mobilization of calcium from the ER. The site of action of CCh for this effect is developmentally regulated. In animals younger than postnatal day 10, the major action of CCh appears to be on mitral cells, enhancing GABA release by reciprocal signaling resulting from increased glutamate release from mitral cells. In animals older than postnatal day 10, CCh appears to modulate transmitter release from dendrites of the interneurons themselves. Our results point to modulation of inhibition as an important role for cholinergic signaling in the OB. Our data also strengthen the emerging idea of a role for store calcium in modulating transmitter release at CNS synapses.

Cyclic nucleotide-gated (CNG) ion channels are central participants in sensory transduction, generating the electrical response to light in retinal photoreceptors and to odorants in olfactory receptors. They are expressed in many other tissues where their specific roles in signaling remain unclear. As is true for many other ion channels, there is a paucity of specific blockers needed to dissect the contributions of these channels to cell signaling. CNG channels are members of the superfamily of voltage-gated ion channels, and the local anesthetic tetracaine is known to block CNG channels in a manner that resembles the block of voltage-gated Na(+) channels. The amine in local anesthetics interacts with the charged selectivity filter of Na(+) channels, while the aromatic ring gets stuck in the inner cavity and has hydrophobic interactions with the residues lining that region. Here we have synthesized a derivative of tetracaine, 3-[(aminopropyl)amino]-N,N-dimethyl-N-(2-[[4-(butylamino)benzoyl]oxy]ethyl)propan-1-aminium acetate (APPA-tetracaine), that contains three positively charged amines at physiological pH instead of one. This compound blocked several different CNG channels in the picomolar to nanomolar concentration range at positive membrane potentials, making it several orders of magnitude more potent than tetracaine. In contrast, significant block of Na(+) channels by APPA-tetracaine required concentrations of hundreds of nanomolar. The results suggest that the highly charged moiety of APPA-tetracaine interacts strongly with the negative charge cluster in the selectivity filter of CNG channels. We propose that a variety of potent and specific ion channel blockers could be generated by expanding on traditional blocker structures to target the selectivity filters of other channels.

Five new tetracaine analogues were synthesized and evaluated for potency of blockade of cyclic nucleotide-gated channels relative to a multiply charged tetracaine analogue described previously (4). Increased positive charge at the tertiary amine end of tetracaine results in higher potency and voltage dependence of block. Modifications that reduce the hydrophobic character at the butyl tail are deleterious to block. The tetracaine analogues described here have apparent affinities for CNGA1 channels that vary over nearly 8 orders of magnitude.

The computational role of the olfactory bulb remains a mystery after 60 yr of physiological research. Recently, Fantana and colleagues proposed a new model of bulb function based on sparse inhibitory connections between glomeruli, the functional units of the bulb, rather than the existing lateral inhibition model. I present a summary of their model here and its implications along with comparison to recent work in the very similar Drosophila olfactory system.

The neural basis of olfactory information processing and olfactory percept formation is a topic of intense investigation as new genetic, optical, and psychophysical tools are brought to bear to identify the sites and interaction modes of cortical areas involved in the central processing of olfactory information. New methods for recording cellular interactions and network events in the awake, behaving brain during olfactory processing and odor-based decision making have shown remarkable new properties of neuromodulation and synaptic interactions distinct from those observed in anesthetized brains. Psychophysical, imaging, and computational studies point to the orbitofrontal cortex as the likely locus of odor percept formation in mammals, but further work is needed to identify a causal link between perceptual and neural events in this area.