Odorant-Dependent Generation of Nitric Oxide in Mammalian Olfactory Sensory Neurons

The gaseous signalling molecule nitric oxide (NO) is involved in various physiological processes including regulation of blood pressure, immunocytotoxicity and neurotransmission. In the mammalian olfactory bulb (OB), NO plays a role in the formation of olfactory memory evoked by pheromones as well as conventional odorants. While NO generated by the neuronal isoform of NO synthase (nNOS) regulates neurogenesis in the olfactory epithelium, NO has not been implicated in olfactory signal transduction. We now show the expression and function of the endothelial isoform of NO synthase (eNOS) in mature olfactory sensory neurons (OSNs) of adult mice. Using NO-sensitive micro electrodes, we show that stimulation liberates NO from isolated wild-type OSNs, but not from OSNs of eNOS deficient mice. Integrated electrophysiological recordings (electro-olfactograms or EOGs) from the olfactory epithelium of these mice show that NO plays a significant role in modulating adaptation. Evidence for the presence of eNOS in mature mammalian OSNs and its involvement in odorant adaptation implicates NO as an important new element involved in olfactory signal transduction. As a diffusible messenger, NO could also have additional functions related to cross adaptation, regeneration, and maintenance of MOE homeostasis

Electrochemical detection of nitric oxide from living cells

Two different types of NO-sensor were developed. The first type of NO-sensor developed was based on the electrochemical entrapment of Ni-TmPyP within a polymer matrix formed by anodic electrodeposition paint on the surface of Pt electrodes. The second type of NO-sensor was developed by polymerizing a layer of Ni-TSPc on a Pt surface followed by layer of Nafion$^\circledR$. The chemical modifications on both types of electrode developed were successfully applied for the sensitive determination of nitric oxide. In the measurement of NO release from living cells, the positioning of the NO-sensor plays an important role. Hence, different procedures were developed for accurate and precise detection of NO. Simultaneous detection of NO and glutamate from cells at a known distance was successfully achieved. Finally, single cell measurement of NO release was achieved with the combination of a developed highly sensitive vibrational disk-shaped NO-sensor and constant distance mode SECM at a sub-$\mu$m distance

Simultaneous detection of L-glutamate and nitric oxide from adherently growing cells at known distance using disk shaped dual electrodes

An ex vivo system for simultaneous detection of nitric oxide (NO) and L-glutamate using integrated dual 250 gin platinum disk electrodes modified individually with suitable sensing chemistries has been developed. One of the sensors was coated with an electrocatalytic layer of Ni tetrasulfonate phthalocyanine tetrasodium salt (Ni-TSPe) covered by second layer of Nation, which stabilises on the one hand the primary oxidation product NO+ and prevents interferences from negatively charged compounds such as NO2-. For glutamate determination, the second electrode was modified with a crosslinked redox hydrogel consisting of Os complex modified poly(vinylimidazol), glutamate oxidase and peroxidase. A manual x-y-z micromanipulator on top of an inverted optical microscope was used to position the dual electrode sensor at a defined distance of 5 mu m from a cell population under visual control. C6 glioma cells were stimulated simultaneously with bradykinin or VEGF to release NO while KCl was used to invoke glutamate release. For evaluation of the glutamate sensors, in some experiments HN10 cells were used. To investigate the sensitivity and reliability of the system, several drugs were applied to the cells, e.g. Ca2+-channel inhibitors for testing Ca2+-dependence of the release of NO and glutamate, rotenone for inducing oxidative stress and glutamate antagonists for analysing glutamate release. With these drugs the NO and glutamate release was modulated in a similar way then expected from previously described systems or even in-vivo measurements. We therefore conclude that our system is suitable to analyse stress-induced mechanisms in cell lines. (c) 2006 Elsevier B.V All rights reserved

Geraniol-evoked electro-olfactogram recordings from olfactory epithelia show significant differences in adaptation for wild-type compared to eNOS-deficient mice.

<p><i>A,</i> Representative recordings of odor-responses to repetitive application of geraniol (1 second duration; 4 seconds interstimulus interval) in wild-type (black) and eNOS deficient mice (red). <i>B,</i> Histogram of mean amplitudes of consecutive responses normalized to the amplitude of the first geraniol response in a stimulus series of wild-type (black bars) and eNOS deficient mice (red). <i>C</i>, Histogram of mean amplitudes of consecutive responses normalized to the amplitude of the first geraniol response in a stimulus series of wild-type olfactory epithelium incubated with Ringer's solution (dark grey bars) or L-NMMA (100 µM, light grey bars). Please notice that incubation of the OE with Ringer`s solution or drug in general led to slower adaptation kinetics, but comparison of the two conditions still shows a significant difference. <i>D,</i> Representative recordings of odor responses to geraniol pulses (2 seconds duration; 28 seconds interstimulus interval). <i>E,</i> Mean amplitude of the second geraniol response normalized to the amplitude of the first response.</p

mRNA of the endothelial isoform of NO-synthase (eNOS) is expressed in olfactory sensory neurons.

<p><i>A,</i> RT-PCR analysis of 1500 purified OSNs with primers specific for eNOS and Gα_olf. <i>B,</i><i> In situ</i> hybridization of eNOS-specific anti-sense and sense probes to cryosections of the murine olfactory epithelium. The scale bars represent 20 µm.</p

Wild-type and eNOS-deletion mutant mice show no differences in amplitude and in decay kinetics of responses to single pulses of geraniol (1 s duration).

<p>(A) Absolute (open squares) and mean amplitudes (filled diamonds) of geraniol responses. Mean values were 1.82±0.17 mV for wild-type and 1.87±0.27 mV for eNOS deletion mutants. (B) Decay kinetics measured as curve width at 50% of the response amplitude. Mean values were 2.49±0.13 s for wild-type and 2.52±0.19 s for eNOS deletion mutants. Error bars indicate SEM.</p

Quantification of BrdU-labeled basal cells in the olfactory epithelium of wild-type and eNOS deficient mice shows no difference in numbers of proliferating cells.

<p>(A) Schematic drawing of a mouse head with the three coronal section levels (referred to as 1, 2 and 3) that were used for quantification of BrdU positive cells. Scheme adapted from A. Puche (<a href="http://www.apuche.org" target="_blank">www.apuche.org</a>). (B) Reconstructions of coronal sections from these section levels and their respective localization (1, 2 and 3). (C) Exemplary anti-BrdU immunofluorescence from the nasal septum of wild-type (WT) and deletion mutant (delMu) mice. Dashed lines indicate the olfactory epithelium. Arrows represent 100 µm. (D) Cell densities of BrdU positive cells of the nasal septum of WT and delMu animals for all three section levels. Error bars indicate SEM.</p

eNOS-protein is localized to somata, dendrites and olfactory knobs but not to the cilia of mature OSNs.

<p><i>A,</i> eNOS immunostaining of cryosections of the olfactory epithelium of OMP-GFP mice. Pictures show endogenous GFP-fluorescence of OSNs (green), eNOS-specific immunostaining (red) as well as the overlay of the endogenous fluorescence and the immunostaining. <i>B,</i> Control without primary antibody. <i>C,</i> Double immunostaining of GFP-positive OSNs with antibodies for eNOS and adenylyl cyclase type III. Pictures show endogenous GFP-fluorescence of OSNs (green), eNOS-specific immunofluorescence (red), adenylyl cyclase type III (blue) and the overlay. The scale bars represent 20 µm.</p