WO2010018545A1 - Détection et utilisation de phéromones - Google Patents

Détection et utilisation de phéromones Download PDF

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WO2010018545A1
WO2010018545A1 PCT/IB2009/053541 IB2009053541W WO2010018545A1 WO 2010018545 A1 WO2010018545 A1 WO 2010018545A1 IB 2009053541 W IB2009053541 W IB 2009053541W WO 2010018545 A1 WO2010018545 A1 WO 2010018545A1
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alarm
inhibitor
pheromones
alarm pheromone
pheromone
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PCT/IB2009/053541
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English (en)
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Julien BRECHBÜHL
Magalie Klaey
Marie-Christine Broillet
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Universite De Lausanne
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4727—Calcium binding proteins, e.g. calmodulin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/30—Psychoses; Psychiatry
    • G01N2800/301—Anxiety or phobic disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/40—Disorders due to exposure to physical agents, e.g. heat disorders, motion sickness, radiation injuries, altitude sickness, decompression illness

Definitions

  • the present invention relates to a bioassay method for testing and identifying alarm pheromones in mammals. Alarm pheromones identified through said method are also described.
  • the olfactory system provides sensory information about the chemical composition of the external world. It fulfils a variety of tasks including locating and evaluating food, reproduction, predator avoidance and social interactions (1). This functional diversity is reflected by the presence of several sensory structures or subsystems within the nasal cavity that are morphologically and molecularly distinct.
  • the main olfactory epithelium occupies the dorsal-posterior region of the nasal cavity and lines the turbinates and parts of the nasal septum. Its main function is the detection and recognition of odorants.
  • the vomeronasal organ (VNO) is critical for the detection of pheromones (2). Pheromones are chemical signals that elicit specific behavioral and physiological responses in recipients of the same species.
  • Axons of the olfactory sensory neurons innervate glomeruli of the main olfactory bulb (MOB), whereas axons of vomeronasal sensory neurons (VSNs) project to the accessory olfactory bulb (AOB).
  • the septal organ (SO) is an island of sensory epithelium on the nasal septum positioned at the nasoplatine duct; it is considered to have a dual function.
  • the most recently discovered olfactory subsystem the so-called Grueneberg ganglion (GG), is present at the tip of the nose; it has a very specific chemosensory function in recognizing alarm pheromones (3).
  • the presence of distinct olfactory subsystems may be one of the features determining the enormous chemosensory capacity of the sense of smell.
  • the Grueneberg Ganglion (GG) is an organ that was first described by Grueneberg in 1973, as a 'ganglion' of unknown function (4). With the limited histological methods available at the time, Grueneberg concluded that this ganglion-like cell mass might belong to the complex of the Nervus terminalis. These cells were "rediscovered" in 2005-2006 (5, 6), thanks to the inspection of whole-mount specimens from one particular gene-targeted mouse strain (7, 8) called OMP-GFP. These mice express GFP as a histological reporter under the control of the OMP promoter.
  • OMP olfactory marker protein (9)
  • the GG is a densely packed population of round cell bodies arranged in an arrowhead structure of 750-1000 ⁇ m in length.
  • the paired organ is located in a shallow depression of the cartilage covered by a dense network of blood vessels.
  • Development of the GG starts around embryonic day 16 and appears to be complete at birth; cell numbers then undergo a minor decrease during postnatal development (5, 6, 10-12). From 300 to 500 cells can be found in each GG of an adult mouse.
  • GG cells are not arranged in a pseudostratified epithelium but organized into tightly packed individual cell clusters, which are usually not thicker than one or two cell layers. Under confocal microscopy, no sustentacular or basal cells are apparent by morphological criteria and GG cells appear to lack dendritic structures or cilia (5, 6, 10-12).
  • GG neurons These processes, which are primary cilia, are the putative site of chemosensory transduction.
  • the GG was assessed for chemosensory receptor expression by extensive RT-PCR and in situ hybridization analyses. Odorant receptors (ORs and TAARs) were found to be only expressed by very few cells during prenatal and perinatal stages (13); a similar number of cells express adenylyl cyclase type III and Golf/s, characteristic signaling elements of the MOE (14).
  • a VNO receptor a distinct subtype of the V2R class, the V2R83 was found to be expressed in the majority of OMP-positive GG neurons.
  • GG neurons express the G proteins Gao and Gai, both of which are also present in sensory neurons of the VNO (13, 15).
  • Mouse GG neurons also express the cGMP-associated signaling proteins phosphodiesterase 2a, cGMP-dependent kinase II, and cyclic nucleotide- gated channel subunit A3 coupled to a chemoreceptor repertoire of cilia-localized particulate guanylyl cyclases (pGC-G and pGC-A) (16).
  • pGC-G and pGC-A cilia-localized particulate guanylyl cyclases
  • APs Alarm pheromones
  • APs are widely used throughout the plant and animal kingdoms. Species such as fish, insects, and mammals signal danger to conspecifics by releasing volatile alarm molecules. Thus far, neither the chemicals, their bodily source, have been isolated or identified in mammals.
  • This object has been achieved by providing a bioassay method for testing and identifying alarm pheromones in mammals comprising the steps of i) contacting an alarm pheromone candidate to Grueneberg ganglion (GG), and ii) detecting via an indicator whether said contact induces an activation of the GG thus indicating that said an alarm pheromone candidate is an alarm pheromone.
  • GG Grueneberg ganglion
  • a further object of the present invention is to provide an alarm pheromone obtained in accordance with the bioassay method of the invention.
  • Another object of the invention is the use of an alarm pheromone of the invention against an animal attacking humans inducing freezing or running away of the attacking animal, as a pest control or to disperse crowd.
  • Fig. 1 Ciliary processes are present on mouse Grueneberg ganglion cells.
  • A Sagital section through the nasal cavity of an OMP (olfactory marker protein)-GFP gene-targeted mouse.
  • OMP olfactory marker protein
  • the intrinsic GFP fluorescence allows the visualization of the different olfactory subsystems: the main olfactory epithelium (MOE) contains neurons that project to glomeruli in the main olfactory bulb (MOB).
  • Neurons of the septal organ (SO) project to the ventral olfactory bulb. Projections of axons from neurons in the vomeronasal organ (VNO) go to the accessory olfactory bulb (AOB).
  • MOE main olfactory epithelium
  • SO septal organ
  • VNO vomeronasal organ
  • AOB accessory olfactory bulb
  • the Grueneberg ganglion (GG) is located underneath the dorsal cartilage at the tip of the nasal cavity (white rectangle), projecting axons to necklace glomeruli in the anterior AOB.
  • D Maximum intensity projection of a GG cell cluster obtained by confocal microscopy (12 ⁇ m).
  • E Scanning electron micrographs of a coronal section of the tip of the nose (line in B).
  • Grapes of GG cells are located along the Se underneath the keratinized epithelium (KE) lining the nasal cavity (NC), (square detailed in F).
  • F Close-up view of the GG cells laying in a fibroblast meshwork (Fb), (square detailed in G).
  • G From the oval cell body of a GG cell (false color green), an axon (false color red) departs to run along the septum; long and thin cilia (false color blue) could be observed.
  • Scale bars are in (A), 1 mm; 0.1 mm (B); 0.25 mm (C); 25 ⁇ m (D); 20 ⁇ m (E-F); 5 ⁇ m (G).
  • Fig. 2 The Grueneberg ganglion is composed of cells of different origins.
  • A TEM micrograph confirming the presence of ensheathing cells (EC), in false color blue, individually packaging GG cells (GC). Ensheathing cells had a triangular cell body, a round nucleus and long arms circling the GC.
  • B-G High-power views of neuronal or glial protein immunoreactivity. The GG cells were recognizable by their OMP-GFP expression (B and E).
  • C The glial marker SlOO ⁇ was expressed in cells surrounding the GG cell clusters.
  • D is the merge image of (B) and (C). White arrow-heads indicate the position of GFP-negative glial cells (A-C).
  • G is the merged image of (E) and (F) where colocalisation appears in yellow.
  • D and E GG cells are located in between the nasal septum (Se) and the keratinized epithelium (KE). Scale bars are in (A), 2 ⁇ m; 20 ⁇ m (B- G).
  • A GFP-labeled GG cells in a coronal slice of tissue from an OMP-GFP mouse. GG cells are lining the nasal septum (Se) and are separated from the nasal cavity (NC) by the keratinized epithelium (KE).
  • B Uptake of the calcium-sensitive dye FURA-2AM into GG cells within the white rectangle from (A) measured at 380 nm before (B) and at the peak of the intracellular calcium increase induced by a pulse of KCl (100 mM) (C).
  • Mc mouse milk
  • NW mammary secretions from lactating female mice
  • NW nipple wash, NW
  • E a mix of odors (Mix) (durene, eugenol, myrtenal, cineole, citral, 4- metylanisol, fenchone at 10 "6 M) and of known mouse pheromones (is
  • FIG. 4 Alarm pheromone sensing depends on a functional Grueneberg ganglion.
  • A-B A statistically significant increase in freezing behavior and a decrease in walking distance is induced by the presence of alarm pheromones (AP) in the ACSF solution (white bars: ACSF; grey bars: ACSF+AP).
  • C Axonal lesions inducing the complete degeneration of the Grueneberg ganglia
  • C Representative tracking of the walking distance covered by a mouse in the plexiglas test chamber in the absence (white square) or in the presence (grey square) of AP in the ACSF container (square).
  • Red dots indicate the position of the mouse at the beginning of the 3-min test session. Axotomy prevent the detection of AP.
  • A-B Pheromonal effect was verified with Student's paired t test and is indicated above bars. Control (Ctrl) and axotomized (Axo) groups were compared with Student's unpaired t test (dotted lines).
  • A-B, E Significance levels are indicated as follow: *P ⁇ 0.05; ns : not significant. Scale bars are in (C), 500 ⁇ m.
  • Fig. 5 Representative whole-cell patch-clamp recordings from a GG neuron and from a MOE neuron (P6).
  • a current-clamp protocol A
  • the GG neuron shows spontaneous spiking activity, while the olfactory neuron does not.
  • Two types of spiking activities can also be observed in this GG neuron, single spikes or multiple spikes (inset in A).
  • a voltage-clamp configuration B
  • spontaneous current variations are recorded in a GG cell.
  • Fig. 6 Induced electrical activity in a representative whole-cell patch-clamp recording from a GG neuron and from a MOE neuron.
  • A Typical current-clamp responses to current injections of 10-40 pA for 500 ms at testing potential (-7OmV). The adapting response in firing frequency following increased current injections can be clearly seen in GG neurons. In contrast, for the olfactory neurons, an increase in spiking activity can be observed with the increase in current injection. No adaptation occurs.
  • B Typical voltage-clamp responses to voltage step of -100 to -20 mV. Large inward and outward currents can be seen in GG neurons compared to MOE neurons.
  • C Mean number of elicited action potentials plotted as a function of the amplitude of the injected currents. Values are expressed as Means ⁇ SE
  • Fig. 7 Schematic representation of a preferred embodiment of the alarm pheromone (AP) collecting device of the invention. Stressed mice are present in an enclosure, for example a closed plexiglas chamber, and release APs (circles). APs are collected on a solid phase micro extraction (SPME) fibre for identification via a GC-MS protocol, and/or APs are directly transferred via the compressed air circulation and bubbling into water or ACSF for further behavioural or physiological testing.
  • SPME solid phase micro extraction
  • the present invention relates to a bioassay method for testing and identifying alarm pheromones in mammals comprising the steps of i) contacting an alarm pheromone candidate to Grueneberg ganglion (GG), ii) detecting via an indicator whether said contact induces an activation of the GG thus indicating that said compound is an alarm pheromone.
  • GG Grueneberg ganglion
  • the GG has a unique and basic morphology; a ganglion protected from the external world by water-permeant keratin.
  • the GG acts as a warning system dedicated to the recognition of short-lived molecules encoding danger. These molecules require immediate attention.
  • the unusual location of the GG at the tip of the nose, far from the main olfactory system, allows the stimuli to be rapidly detected.
  • AP sensing is a conserved modality present from primitive organisms such as worms to humans.
  • the presence of a GG has been identified in all mammalian species looked at so far including human embryos.
  • a “mammal” refers to a rodent, canine, feline, bovine, equine, porcine and other livestock, deer, goats, sheep and human and non-human primates.
  • the alarm pheromone candidate that may activate the GG in the present invention is selected from the group comprising a pheromone molecule, an odorant molecule and/or inhaled molecule.
  • said alarm pheromone candidate is a natural chemical, a synthetic chemical, a natural peptide or a synthetic peptide, a volatile and water-soluble chemical molecule.
  • the alarm pheromone candidate to be tested will be isolated and collected by any isolation system known by those skilled in the art (see for example Jiang et al., 1989) (3).
  • the method described in the present application will be used to isolate and collect the alarm pheromone candidate (Fig. 7).
  • the alarm pheromone candidate isolated according to one of the techniques know in the art or according to the method described and claimed in the present invention is contacted with a GG in any manner designed to deliver said compound to the GG.
  • a GG is comprised in coronal slice of brain tissue
  • this is done by applying a composition comprising said alarm pheromone candidate to said coronal slice of tissue.
  • Applicants have developed an acute tissue preparation enabling measuring, monitoring or detecting the chemical or electrical activity of a mice neuron GG (see example 2). With this preparation, Applicants are able to measure, monitor or detect the chemical or electrical activity, on the same specimen, from GG cells and from main olfactory neurons. This preparation has the advantage of maintaining the native cellular topography of the GG.
  • the method of determining the activity or activation of the GG or the activity or activation of the GG neuron in a mammal would be to monitor in vivo the behavior of the animal in response to the alarm pheromone candidate, then the said alarm pheromone candidate, or a composition containing said alarm pheromone candidate, will be administered to the mammal either by local injection, inhalation, local application or ingestion.
  • the alarm pheromone candidate will be administered to the mammal by inhalation or by local application e.g. deposited on the tip of the nose of the mammals.
  • the method comprises detecting via an indicator whether said contact induces an activation of the GG thus indicating that said compound is an alarm pheromone candidate.
  • the indicator of the present invention indicates, shows or signals that the contact of the alarm pheromone candidate with, for example a receptor found on the GG or an intracellular messenger, induces an activation of the neuronal activity of said GG thus indicating that said compound is an alarm pheromone candidate.
  • Detecting an activation of a neuronal activity of said GG for the present invention can be accomplished by any means known in the art.
  • One method of measuring, monitoring or detecting the activity of a neuron includes detection or measurements of changes in action potentials, membrane potentials and receptor potentials compared to the resting membrane potential of a neuron.
  • Other methods of detecting the activity of a neuron include measuring or detecting the propagation of an action potential from one neuron to the next, or measuring or detecting the voltage or ion influx/efflux along the neuron, or measuring or detecting the release of neurotransmitters into a synaptic cleft.
  • Methods of determining or measuring activity of neurons are well known in the art and include, for example, patch clamps, monitoring levels of movement of labeled neurotransmitters, e g , radioactively labeled neurotransmitters.
  • MRI BOLD blood oxygen level dependent contrast
  • monitoring metabolism and metabolic breakdown products monitoring glucose intake, monitoring Ca 2+ movement, monitoring expression of IEGs (immediate early genes) such as, but not limited to, cFos, Erg- 1 , ZENK and Arc.
  • IEGs immediate early genes
  • Methods of determining neuronal activity are disclosed in Principles of Neural Science, 41h Edition, Kandel. E. R., et al (Eds). McGraw-Hill. 2000 and The Neuron Cell and Molecular Biology, 3rd Edition, Lovitan. LR. , and Kachmarek. L. K., Oxford University Press, New York, New York, 2001 both of which are incorporated by reference herein.
  • Additional methods of measuring the activity of neurons include measuring, detecting or monitoring behavioral responses in an animal.
  • the Applicants have reported in Brechb ⁇ hl et al. that severance of the GG axonal projections abrogates the "freezing response'" in mice that is normally associated with the detection of alarm pheromones.
  • one method of determining the "activity of the GG" or the ""activity of the GG neuron" in a mammal would be to monitor the behavior of the mammal in response to the alarm pheromone candidate.
  • the behavior that is monitored in the mammal should be well- correlated to the activation of the neurons or circuitry in question.
  • Additional methods of measuring or detecting the activity of a neuron include monitoring second messengers within the cells.
  • second messengers include, but are not limited to, calcium ions (both from external sources and internal stores), cyclic GMP (cGMP) production or breakdown, cyclic AMP (cAMP) production or breakdown, generation or breakdown of inositol triphosphate (IP3), generation or breakdown of diacyglycerides (DAGs) and even gene transcription in response to activation or inhibition of transcription factors.
  • cGMP cyclic GMP
  • cAMP cyclic AMP
  • IP3 inositol triphosphate
  • DAGs diacyglycerides
  • Numerous methods in the art exist for the detection and measurement of second messengers that can be used as direct or indirect measurements of the neuron activity.
  • transcription levels can be measured to assess the effects of an alarm pheromone candidate on signal transduction.
  • the indicator is selected from the group comprising a chemical indicator, a genetically encoded indicator, an action potential, a membrane potential, a receptor potentials, a voltage or ion influx/efflux along the neuron, or a release of neurotransmitters into a synaptic cleft.
  • the indicator is a chemical indicator such as a small molecule that can chelate calcium ions. All these molecules are based on an EGTA homologue called BAPTA, with high selectivity for calcium (Ca2+) ions versus magnesium (Mg2+) ions.
  • BAPTA EGTA homologue
  • Non-limiting examples of indicators include fura-2, indo-1, fluo-3, Calcium Green- 1. These dyes are generally used with the chelator carboxyl groups masked as acetoxymethyl esters, in order to render the molecule lipophilic and to allow easy entrance into the cell. Once the indicator is in the cell, cellular esterases will free the carboxyl and the indicator will be able to bind calcium.
  • genetically encoded indicators such as for example fluorescent proteins derived from green fluorescent protein (enhanced GFP) or its variants (e.g. circularly permuted GFP, YFP, CFP), fused with calmodulin (CaM) and the M 13 domain of the myosin light chain kinase, which is able to bind CaM.
  • GFP green fluorescent protein
  • CaM calmodulin
  • Genetically encoded indicators do not need to be loaded onto cells, instead the genes encoding for these proteins can be easily transfected to cell lines. It is also possible to create transgenic animals expressing the dye in all cells or selectively in certain cellular subtypes.
  • Non- limiting examples of genetically encoded indicators are Pericams (circularly permuted GFPfused to calmodulin and its target peptide, M13, see Nagai et al., 2001) and Cameleons (PremoTM Cameleon Calcium Sensor, a non-organic dye indicator for intracellular calcium signal measurements, Invitrogen).
  • alarm pheromones are only released when a predator damages the skin of the animal. Indeed alarm pheromones are located in "club cells" that have no contact with the external environment; the pheromone is released only when the skin of the animal is damaged. Usually, the alarm pheromone is a chemical responsible for danger signaling in mammals.
  • Alarm pheromones or APs are expected to have low molecular weights and to be extremely volatile to allow fast diffusion from the source but a quick decline once the danger has past. These chemical properties make the identification task all the more difficult. In insects, most alarm pheromones have molecular weights of 200-300. Mice and other mammals increase the longevity of small volatile pheromones such as sex pheromones or territorial pheromones in associating them with urinary proteins which release them slowly.
  • the present invention also relates to an alarm pheromone obtained, or obtainable, in accordance with the claimed bioassay method for testing and identifying an alarm pheromone.
  • the bioassay method of the present invention may further comprise the step of contacting said activated GG with an inhibitor. If said contact with an inhibitor induces an inactivation of inhibition of the GG, as assessed by methods of measuring the activity of neurons described before, this further confirms and indicates that said alarm pheromone candidate is an alarm pheromone.
  • the bioassay method of the present invention may further comprise the step of contacting said activated GG with an inhibitor candidate. If said contact with an inhibitor candidate induces an inactivation of inhibition of the GG, as assessed by methods of measuring the activity of neurons described before, this further confirms and indicates that i) said alarm pheromone candidate is an alarm pheromone and ii) that said inhibitor candidate is an inhibitor of the GG.
  • the present invention also considers the use of an alarm pheromone, or of a composition containing said alarm pheromone, of the invention against an animal attacking humans inducing freezing or running away of the attacking animal.
  • the use of said alarm pheromone, or of a composition containing said alarm pheromone, as a pest control or to disperse crowd is also considered.
  • the alarm pheromone will be administered to the animal by inhalation or by local application e.g. on the tip of the nose.
  • other route of administration of the alarm pheromone candidate such as oral, subcutaneous, intravenous, intraarterial, intraperitoneal and intramuscular are also contemplated.
  • the alarm pheromone will be administered to the animal by inhalation.
  • one additional aspect of the present invention is to provide a pharmaceutical composition containing an alarm pheromone as active pharmaceutical ingredient.
  • the pharmaceutical agent can be in a variety of well known formulations and administered using any of a variety of well known methods of administration such as intra-nasal, oral, subcutaneous, intravenous, intraarterial, intraperitoneal and/or intramuscular are also contemplated. or the like. Non-injectable formulations are preferred and intra-nasally or inhaled formulations are particularly preferred.
  • compositions adapted for nasal administration wherein the pharmaceutically acceptable carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, e.g, by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • suitable compositions wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.
  • compositions adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers or insufflators.
  • the formulation may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, among others.
  • suspending agents as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, among others.
  • Useful intranasal formulations of an alarm pheromone may contain a stabilizers and a surfactants.
  • polyoxyethylene castor oil derivatives such as polyoxyethylene-glycerol-triricinoleate, also known as polyoxyl 35 caster oil (CREMOPHOR EL), or poloxyl 40 hydrogenated castor oil (CREMOPHOR RH40) both available from BASF Corp.
  • mono-fatty acid esters of polyoxyethylene (20) sorbitan such as polyoxyethylene (20) sorbitan monolaurate (TWEEN 80), polyoxyethylene monostearate (TWEEN 60), polyoxyethylene (20) sorbitan monopalmitate (TWEEN 40), or polyoxyethylene 20 sorbitan monolaurate (TWEEN 20) (all available from ICI Surfactants of Wilmington, Del.); polyglyceryl esters, such as polyglyceryl oleate; and polyoxyethylated kernel oil (LABRAFIL, available from Gattefosse Corp.).
  • polyoxyethylene castor oil derivatives such as polyoxyethylene-glycerol-triricinoleate, also known as poly
  • the surfactant will be between about 0.01% and 10% by weight of the pharmaceutical composition.
  • the pharmaceutically useful stabilizers are antioxidants such as sodium sulfite, sodium metabisulfite, sodium thiosulfate, sodium formaldehyde sulfoxylate, sulfur dioxide, ascorbic acid, isoascorbic acid, thioglycerol, thioglycolic acid, cysteine hydrochloride, acetyl cysteine, ascorbyl palmitate, hydroquinone, propyl gallate, nordihydroguaiaretic acid, butylated hydroxytoluene, butylated hydroxyanisole, alpha-tocopherol and lecithin.
  • antioxidants such as sodium sulfite, sodium metabisulfite, sodium thiosulfate, sodium formaldehyde sulfoxylate, sulfur dioxide, ascorbic acid, isoascorbic acid, thioglycerol, thiogly
  • the stabilizer will be between about 0.01% and 5% by weight of the pharmaceutical composition.
  • Suspensions may also include chelating agents such as ethylene diamine tetraacetic acid, its derivatives and salts thereof, dihydroxyethyl glycine, citric acid and tartaric acid among others. Additionally, proper fluidity of a suspension can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants, such as those previously mentioned.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active pharmaceutical ingredient may be mixed with at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants such as glycerol; (d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (e) solution retarding agents such as paraffin; (f) absorption accelerators such as quaternary ammonium compounds; (g) wetting agents such as cetyl alcohol and glycerol monostearate;(h) absorbents such as kaolin and bentonite clay; and (i) lubric, such as sodium cit
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, capsules, pills and granules can be prepared with coatings and shells such as enteric coating and other coatings well-known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition such that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
  • Examples of embedding compositions which can be used include polymeric substances and waxes.
  • capsules or tablets slowly releasing the compound in the environment useful in the case of pest control.
  • Liquid dosage forms for oral administration or for spray formulation include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsif ⁇ ers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof.
  • inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emuls
  • This invention also envisages the use of alarm pheromone in a pharmaceutically acceptable salt form.
  • salts may include sodium, potassium, calcium, aluminum, gold and silver salts.
  • salts formed with pharmaceutically acceptable amines such as ammonia, alkyl amines, hydroxyalkylamines, N-methylglucamine and the like.
  • Certain basic compounds also form pharmaceutically acceptable salts, e.g., acid addition salts.
  • pyrido-nitrogen atoms may form salts with strong acid, while compounds having basic substituents such as amino groups also form salts with weaker acids.
  • suitable acids for salt formation are hydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicylic, malic, fumaric, succinic, ascorbic, maleic, pamoic, methanesulfonic and other mineral and carboxylic acids well known to those skilled in the art.
  • the salts are prepared by contacting the free base form with a sufficient amount of the desired acid to produce a salt in the conventional manner.
  • the free base forms may be regenerated by treating the salt with a suitable dilute aqueous base solution such as dilute aqueous NaOH, potassium carbonate, ammonia and sodium bicarbonate.
  • a suitable dilute aqueous base solution such as dilute aqueous NaOH, potassium carbonate, ammonia and sodium bicarbonate.
  • the free base forms differ from their respective salt forms somewhat in certain physical properties, such as solubility in polar solvents, but the acid and base salts are otherwise equivalent to their respective free base forms for purposes of the invention.
  • the alarm pheromone as active pharmaceutical ingredient is present in an amount between about 0.001% and 20% by weight of the pharmaceutical composition.
  • the present invention also contemplates a bioassay method for testing and identifying an inhibitor to a Grueneberg ganglion in mammals comprising the steps of i) contacting an alarm pheromone to a GG which is able to activate said GG, ii) simultaneously or subsequently contacting an inhibitor candidate to said GG, iii) and detecting via an indicator whether said contact induces an inactivation or inhibits an activation of the GG by the alarm pheromone able to activate said GG thus indicating that said an inhibitor candidate is an inhibitor or a competitive inhibitor to an alarm pheromone.
  • an inhibitor candidate refers to a compound that inactivates or inhibits a GG previously activated by an alarm pheromone.
  • said inhibitor candidate is selected from the group comprising a natural chemical, a synthetic chemical, a natural peptide or a synthetic peptide.
  • Non-limiting examples include also gaseous molecules, ions and metallic molecules.
  • the indicator of the present invention indicates, shows or signals that the contact of an inhibitor candidate with, for example a receptor on the GG, induces an inactivation or activation inhibition of the neuronal activity of said GG thus indicating that said compound is an inhibitor candidate.
  • Detecting an inactivation or activation inhibition of a neuronal activity of said GG for the present invention can be accomplished by any means known in the art.
  • One method of measuring, monitoring or detecting the inactivation or activation inhibition of a neuron includes detection or measurements of changes in action potentials, membrane potentials and receptor potentials compared to the membrane potential of an activated neuron.
  • Other methods of detecting the activity of a neuron include measuring or detecting the loss or decrease of the propagation of an action potential from one neuron to the next, or measuring or detecting the loss or decrease of the voltage or ion influx/efflux along the neuron, or measuring or detecting the loss or decrease of release of neurotransmitters into a synaptic cleft.
  • Methods of determining or measuring activity of neurons are well known in the art and include, for example, patch clamps, monitoring levels of movement of labeled neurotransmitters, e g , radioactively labeled neurotransmitters.
  • MRI BOLD blood oxygen level dependent contrast
  • monitoring metabolism and metabolic breakdown products monitoring glucose intake, monitoring Ca 2+ movement, monitoring expression of IEGs (immediate early genes) such as, but not limited to, cFos, Erg- 1 , ZENK and Arc.
  • Additional methods of measuring the activity of neurons include measuring, detecting or monitoring behavioral responses in an animal.
  • the Applicants have reported in Brechb ⁇ hl et al. that severance of the GG axonal projections abrogates the "freezing response'" in mice that is normally associated with the detection of alarm pheromones.
  • one method of determining the activity or activation of the GG or the activity or activation of the GG neuron in a mammal would be to monitor the behavior of the mammal in response to the inhibitor candidate.
  • the behavior that is monitored in the mammal should be well- correlated to the inactivation or activation inhibition of the neurons or circuitry in question.
  • Additional methods of measuring or detecting the activity of a neuron include monitoring second messengers within the cells.
  • second messengers include, but are not limited to, calcium ions (both from external sources and internal stores), cyclic GMP (cGMP) production or breakdown, cyclic AMP (cAMP) production or breakdown, generation or breakdown of inositol triphosphate (IP3), generation or breakdown of diacyglycerides (DAGs) and even gene transcription in response to activation or inhibition of transcription factors.
  • cGMP cyclic GMP
  • cAMP cyclic AMP
  • IP3 inositol triphosphate
  • DAGs diacyglycerides
  • Numerous methods in the art exist for the detection and measurement of second messengers that can be used as direct or indirect measurements of the neuron activity.
  • transcription levels can be measured to assess the effects of the inhibitor candidate on signal transduction.
  • the indicator is selected from the group comprising a chemical indicator, an action potential, a membrane potential, a receptor potential, a voltage or ion influx/efflux along the neuron, or a release of neurotransmitters into a synaptic cleft.
  • the indicator is a chemical indicator such as a small molecule that can chelate calcium ions. All these molecules are based on an EGTA homologue called BAPTA, with high selectivity for calcium (Ca2+) ions versus magnesium (Mg2+) ions.
  • Non-limiting examples of indicators include fura-2, indo-1, fluo-3, Calcium Green- 1.
  • These dyes are generally used with the chelator carboxyl groups masked as acetoxymethyl esters, in order to render the molecule lipophilic and to allow easy entrance into the cell. Once the indicator is in the cell, cellular esterases will free the carboxyl and the indicator will be able to bind calcium.
  • genetically encoded indicators such as for example fluorescent proteins derived from green fluorescent protein (enhanced GFP) or its variants (e.g. circularly permuted GFP, YFP, CFP), fused with calmodulin (CaM) and the M 13 domain of the myosin light chain kinase, which is able to bind CaM.
  • GFP green fluorescent protein
  • CaM calmodulin
  • Genetically encoded indicators do not need to be loaded onto cells, instead the genes encoding for these proteins can be easily transfected to cell lines. It is also possible to create transgenic animals expressing the dye in all cells or selectively in certain cellular subtypes.
  • Non- limiting examples of genetically encoded indicators are Pericams (circularly permuted GFPfused to calmodulin and its target peptide, M13, see Nagai et al., 2001) and Cameleons (PremoTM Cameleon Calcium Sensor, a non-organic dye indicator for intracellular calcium signal measurements, Invitrogen).
  • the present invention also considers that the inhibitor can be a competitive inhibitor.
  • the inhibitor acts on the same active site as the alarm pheromone.
  • the alarm pheromone cannot bind for example the receptor on the GG while the inhibitor is there.
  • Characteristic for this mode of inhibition is that increasing the concentration of competitive inhibitor reduces the effect of alarm pheromone.
  • the present invention also relates to an inhibitor or a competitive inhibitor obtained, or obtainable, in accordance with the claimed bioassay method for testing and identifying an inhibitor.
  • the present invention also considers the use of an inhibitor or a competitive inhibitor of the invention for handling mammals in stress situation.
  • the inhibitor or a competitive inhibitor will be administered to the animal by inhalation or by local application e.g. at the tip of the nose at the rostral portion of the inside of the nostrils.
  • other route of administration of the alarm pheromone candidate such as oral, subcutaneous, intravenous, intraarterial, intraperitoneal and intramuscular are also contemplated.
  • the inhibitor or a competitive inhibitor will be administered to the animal by inhalation.
  • one additional aspect of the present invention is to provide a pharmaceutical composition containing an inhibitor or the competitive inhibitor as active pharmaceutical ingredient.
  • the pharmaceutical agent can be in a variety of well known formulations and administered using any of a variety of well known methods of administration such as intranasal, oral, subcutaneous, intravenous, intraarterial, intraperitoneal and intramuscular are also contemplated. or the like. Non-injectable formulations are preferred and intra-nasally or inhaled formulations are particularly preferred.
  • the inhibitor or the competitive inhibitor as active pharmaceutical ingredient is present in an amount between about 0.001% and 20% by weight of the pharmaceutical composition.
  • the present application also concerns a method for collecting an alarm pheromone, or an alarm pheromone candidate, from a mammal comprising the steps of i) transferring said mammal into a closed area, ii) applying a stress situation to said mammal so as to induce the secretion of an alarm pheromone or an alarm pheromone candidate, iii) and recovering the secreted and volatile alarm pheromone or an alarm pheromone candidate.
  • alarm pheromones are chemicals responsible for danger signaling in mammals, they have low molecular weights and are extremely volatile to allow fast diffusion from the source but have quick decline once the danger has past.
  • the stress situation is induced by applying, for example, a CO2 inhalation, electric shocks, cold stress, restriction stress, forced swimmer test or a combination of one or more of these situations.
  • this stress induces mammals, in particular mice, to release volatile and water-soluble alarm pheromones or compounds of interest which are then collected via tubing into ACSF.
  • Alarm pheromones or compounds of interest may also be collected by using, for example, one of the following detection or collection system.
  • MS mass spectrometry
  • FIR Fourier transform infrared spectroscopy
  • FID flame ionisation detection
  • EAD electroantennographic detection
  • SPME solid phase microextraction
  • PDMS polydimethylsiloxane
  • an adsorptive one e.g. carboxen (CAR), carbowax (CW)
  • film thicknesses ranging from 7 to 100 ⁇ m.
  • Analytes are absorbed from water or from head-space samples. After SPME extraction, the analytes can be thermally desorbed into the heated gas chromatograph injection port and analyzed directly by gas chromatography. SPME completely eliminates the use of organic solvents and substantially reduces the amount of sample needed to run an analysis from milliliters to microliters, thus improving analytical conditions when using biological samples from small animals. This last method has been successfully applied to the five most common pheromones studied in mice research.
  • Alarm pheromones or compounds of interest are solubilised, for example, in artificial cerebrospinal fluid (ACSF) solution in accordance with the present invention.
  • This solution is then transferred into a 20 ml headspace vial which is sealed immediately with Teflon-coated butyl septa and a hand crimper for 20 mm seals (Supelco).
  • controls ACSF ACSF with mice general pheromones
  • 30 min old ACSF + alarm pheromones are analyzed.
  • Solid phase microextraction is performed on carboxen/polydimethylsiloxane, carbowax/divinylbenzene and polydimethylsiloxane fibers of different film thickness, respectively (Supelco).
  • a further object of the present invention is to provide a device for collecting alarm pheromones, said device comprising: i) an enclosure for receiving the mammal, ii) means for applying stress to the mammal present in said enclosure, and iii) a collector for collecting the volatile alarm pheromone secreted by the stressed mammal.
  • the enclosure 91 of the device for collecting alarm pheromones is a closed chamber, made for example of Plexiglas, in which the mammal 99, e.g. a mouse, can be introduced through a door that is not represented in the figure for the sake of conciseness.
  • the means for applying stress to the mammal 99 present in the enclosure comprises for example a stress gas inlet for introducing a gas, for example CO 2 , into the chamber and/or electrodes coupled to an electric generator for applying electric shocks to the enclosure 91 and/or a refrigeration circuit for cooling the chamber 91.
  • the stress applied to the mammal 99 is a restriction stress, for example a food restriction stress, for the application of which no specific means is required.
  • the collector for collecting the volatile alarm pheromone secreted by the stressed mammal 99 comprises for example a SPME fiber 92 as described above, which is releasably attached to the enclosure 91 with one extremity 921 at least partly introduced into the closed chamber, so that the SPME fiber 92 collects at least part of the volatile APs present in the atmosphere of enclosure 91.
  • the collector comprises a gas inlet 93 for introducing a compressed gas or gas mixture, for example compressed air, into the enclosure 91, a gas outlet 94 and a fluid receptacle 95 containing a fluid 951, for example water or ACSF, in which the alarm pheromones to be collected are soluble.
  • the gas outlet 94 is configured to duct the gas or gas mixture exiting the enclosure 91 into the fluid receptacle 95, below the surface of the fluid 951.
  • overpressure is generated in the enclosure 91 by introducing for example compressed air through the gas inlet 93, which tends to push the gas mixture constituting the atmosphere of the chamber out of the enclosure 91 through the gas outlet 94.
  • the gas mixture exiting the enclosure 91 typically comprises air and/or the compressed gas introduced into the chamber, and at least part of the alarm pheromones secreted by the stressed mice 99. This gas mixture passes through the fluid 951 in which the soluble APs are solved and thus captured.
  • the collector of the device of the invention is a combination of both embodiments described above.
  • the device of the invention is described above for the collection of alarm pheromones. It can however be used for collecting other volatile substances present in the atmosphere of the enclosure 91.
  • the device of the invention can for example be used for collecting general pheromones (GPs) produced by freely behaving and communicating mammals.
  • GPs general pheromones
  • mice and tissue processing The OMP-green fluorescent protein (GFP) gene-targeted strain of mice has been described previously (28). PO to adult (6 months) heterozygous male and female mice were used. Animal care was in accordance with the Swiss legislation and the local veterinary authority. Mice were killed by CO2 or cervical dislocation.
  • GFP OMP-green fluorescent protein
  • Nasal cavities were prepared in ice-cold artificial cerebrospinal fluid (ACSF), containing NaCl 118 mM, NaHCCb 25 mM, D-Glucose 10 mM, KCl 2 mM, MgCl 2 2 mM, NaH 2 PO 4 1.2 mM, CaCl 2 2 mM (pH 7.4) saturated with oxycarbon gas (95% O 2 : 5% CO 2 ) under a fluorescence-equipped dissecting microscope (Wild MlO, Leica) to localize the Grueneberg ganglion (GG) cells.
  • ACSF ice-cold artificial cerebrospinal fluid
  • Tissue fragments containing fluorescent GG cells were fixed in PAF 4% (4% paraformaldehyde in 0.1 M phosphate buffer) for 5 h and cryoprotected with an overnight incubation in 30% sucrose solution; they were further embedded in Tissue-Tek (OCT, Sakura) and frozen with liquid nitrogen. 12-16 ⁇ m coronal and sagital cryosections were prepared (HM 550, Microm International cryostat) and deposited on gelatin-coated slides (chromium (I ⁇ I)-gelatin from porcine skin, Fluka) (SuperFrost, Menzel-Glaser).
  • the neuronal markers used were NeuN (1 : 100, Monoclonal mouse Anti-Neuronal nuclei, Chemicon) and TUJl (1 :2000, Monoclonal mouse Anti-Neuronal Class III ⁇ -Tubulin, Covance).
  • the glial markers tested were GFAP (1 :250, Polyclonal Rabbit Anti-Glial Fibrillary Acidic Protein,
  • DakoCytomation DakoCytomation
  • SlOO ⁇ 1: 1000, Polyclonal Rabbit Anti-SlOO ⁇ , Swant
  • the marker of the olfactory signal transduction machinery used was G ⁇ i2 (1 :250, Polyclonal Rabbit Anti- G 0112 , Santa Cruz).
  • the cilia markers used were Ac-Tubulin for axonemes (1 :2000, Monoclonal Mouse Anti-Acetylated Tubulin, Sigma) and ⁇ -Tubulin (1 :5000, Polyclonal Rabbit Anti- ⁇ - Tubulin, Sigma) for centrioles.
  • the secondary antibodies used were coupled to Cy3 (Cy3 -conjugated AffmiPure Goat Anti-Mouse/Rabbit/Ginea pig (H+L), Jackson ImmunoResearch) or Cy5 (Cy5-conjugated AffmiPure Goat Anti-Mouse IgG, F(ab') 2 fragment specific, Jackson ImmunoResearch) for double labeling. Immunohistochemistry experiments were performed with a protocol adapted from (29).
  • cryosections were rehydrated in PBS for 5 minutes, post-fixed with PAF 4% for 15 min, and blocked in a PBS solution containing NGS (Normal Goat Serum, Jackson ImmunoResearch Laboratories) 10% and Triton X-100 (Fluka) 0.5%, for 3 h, at room temperature (RT).
  • Primary antibodies were applied to the cryosections for 16 h, at RT.
  • Cryosections were then washed three times with a PBS solution containing NGS 2%, for 5 min each, and then incubated with secondary antibodies (1:200, in PBS containing NGS 2%) for 1 h, at RT.
  • mice were perfused and fixed transcardially with 2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M phosphate buffer.
  • Mouse heads were dissected and placed overnight in the same solution. They were then briefly rinsed in PBS and included in 5% agarose. Coronal and horizontal sections of 80 ⁇ m were obtained with a vibroslicer (VT 100OS, Leica). Selected sections were washed twice in 0.1 M cacodylate buffer for 5 min.
  • TEM Transmitted
  • SEM scanning
  • tissue samples were prepared: the upper nasal cavities and coronal 80 ⁇ m sections of the GG. They were then fixed in 2.0% glutaraldehyde in 0.2 M cacodylate buffer overnight, at 4°C. After rinsing in cacodylate buffer, samples were dehydrated using increasing alcohol concentrations and dried by the critical point method.
  • Acute tissue slices containing GG cells were prepared from 30 min to 6 months-old OMP- GFP mice.
  • the superior anterior part of the mouse head was collected and rinsed in cold ACSF.
  • the tip of the head was embedded in 5% low-melting agar (A7002 Sigma, St. Louis, MO) prepared in Ringer (NaCl 140 mM, Ca 2+ 2 mM, KCl 5 mM, Hepes 10 mM, pH 7.6) and transferred on ice for 30 s for solidification.
  • the agar block was then fixed onto the holder of the vibratome (VT 1200S, Leica). 80 ⁇ m coronal sections of the GG were cut in cold oxygenated ACSF.
  • Odorants and pheromones were prepared as 0.5 mM solutions in dimethyl sulfoxide (DMSO) and kept protected from light. Final solutions were made fresh before each experiment by adding the chemicals to the ACSF solution (pH 7.4). Final DMSO concentrations ( ⁇ 0.1%) had no effect on Ca + concentrations.
  • the odorants used were: amyl acetate, durene, eugenol, myrtenal, cineole, citral, 4-metylanisol, fenchone at 10 6 M.
  • the pheromones used were isobutylamine, 2-heptanone, pentylacetate, dimethylpyrazine, ⁇ / ⁇ -farnesenes at 10 ⁇ 7 M.
  • mice at the time were transferred into a closed Plexiglas chamber (38.5 x 11.5 x 15 cm) and killed by CO 2 inhalation.
  • ACSF containing 100 mM KCl (NaCl 35 mM, NaHCO 3 25 mM, KCl 100 mM, CaCl 2 0.5 mM, MgCl 2 3 mM, NaH 2 PO 4 1.2 mM, glucose 10 mM, saturated with oxycarbon) was used to test cell viability.
  • the nasal cavities of PO and P6 months mice were perfused from the back of the head to the opening of the naris for 15 min with 1 mM Lucifer yellow solution (Fluka) at 37 0 C. After the perfusion, the anterior part of the head was incubated in the same solution for 1 h. Cryosections of 5 ⁇ m were cut, postfixated and counterstained with DAPI for confocal observation (SP5, Leica).
  • Fig. 2A Immunostaining experiments demonstrated the glial origin of the wrapping cells by positive staining for SlOO ⁇ (Fig. 2B-D) and GFAP.
  • a GG comprises two different cell populations: glial cells and neurons (GFP- positive cells) which bear multiple primary cilia, putative sites of chemosensory transduction.
  • GFP- positive cells glial cells and neurons
  • KE keratinized epithelium
  • the keratinized epithelium covering the GG cells was found to be permeant to hydrophilic substances suggesting that water soluble chemostimuli have access to the outside zone of the GG cell clusters where primary cilia are located.
  • a similar morphological situation is observed in C. elegans in which the cilia of the amphid chemosensory neurons (AWA, AWB, AWC), located under the cuticle, sense volatile and hydrophilic odors via direct diffusion (19).
  • GG chemostimulus a calcium-sensitive dye.
  • GG cells were identified by the intrinsic green fluorescence of GFP in their cell bodies (Fig. 3A) and the uptake of the dye was confirmed by fluorescence measurements (Fig. 3B).
  • Chemical stimuli were delivered in oxycarbonated artificial cerebrospinal fluid (ACSF) continuously perfused on the tissue slices in the imaging chamber. Cell viability was assessed by pulses of KCl (Fig. 3C-F).
  • GG cells contrary to olfactory neurons, GG cells, with their primary cilia localized in an extracellular matrix made of type IV collagen and not in a mucus layer can act independently from the calcium concentration present in their environment.
  • the isolated nature of the GG makes it suitable for lesion experiments (axotomy) allowing axonal projection bundles of GG cells to be sectioned (6). After surgery, pups were given back to their mothers and mother-pup recognition was not affected by the procedure.
  • mice from P 1 to P6 are used and killed by cervical dislocation. Heads are dissected on fresh cold oxycarbone Ringer (NaCl 118 mM, NaHCO 3 25 mM, D-Glucose 10 mM, KCl 2 mM, MgCl 2 2 mM, NaH 2 PO 4 1.2 mM, CaCl 2 2 mM, pH 7.4) under a GFP fluorescent binocular lamp (Wild MlO, Leica). Full nasal cavities are extracted from heads by horizontal cutting of the nasal septum and nostrils.
  • the GG cell In a current-clamp protocol (Fig. 5), at resting potential (in this cell -70 mV), the GG cell has a spontaneous spiking activity of 114 ⁇ 19 mV (averaged amplitude), while the olfactory neuron does not.
  • the spiking activity occurs with a frequency of 1.9 ⁇ 0.8 s-1.
  • Two types of spiking activities can also be observed, single spikes or multiple spikes. Single spikes happen at a frequency of 0.3 ⁇ 0.1 s-1 and multiple spikes at 0.7 ⁇ 0.3 s-1. In each group of multiple spikes, a number of 2.5 ⁇ 0.8 spikes can be observed that show adaptation (inset in Fig. 5A).
  • a voltage-clamp configuration Fig. 5B
  • spontaneous current variations are recorded in a GG cell, with an amplitude of 68 ⁇ 18 pA and a frequency of 5.2 ⁇ 0.9 s-1. Values are expressed as Mean ⁇ SE.
  • GG neurons In order to test whether the chemosensitivity of GG cells is preserved, we have investigated the chemosensory properties of GG mouse neurons using electrophysiological measurements on this acute tissue preparation. We want to verify if GG neurons can encode the chemosensory information of alarm pheromones into action potentials that would be transmitted to the olfactory bulb.

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Abstract

La présente invention concerne un procédé de biodosage permettant de rechercher et d'identifier des phéromones d'alarme chez des mammifères. La présente invention concerne également les phéromones d'alarme identifiées par ledit procédé.
PCT/IB2009/053541 2008-08-15 2009-08-11 Détection et utilisation de phéromones WO2010018545A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013138948A1 (fr) * 2012-03-19 2013-09-26 Rugen Therapeutics R & D (Shanghai) Co., Ltd. Protéines de détection de calcium sensibles à l'oxydoréduction et leurs méthodes d'utilisation
EP2772487A1 (fr) * 2013-02-27 2014-09-03 Université de Lausanne Phéromones d'alarme et leur utilisation
CN108168967A (zh) * 2017-12-12 2018-06-15 重庆师范大学 一种鱼类预警信息素的制备方法

Non-Patent Citations (4)

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Title
BRECHBUHL J ET AL: "Grueneberg ganglion cells mediate alarm pheromone detection in mice", SCIENCE 20080822 US,, vol. 321, no. 5892, 22 August 2008 (2008-08-22), pages 1092 - 1095, XP002550064 *
FLEISCHER JOERG ET AL: "Olfactory receptors and signalling elements in the Grueneberg ganglion", JOURNAL OF NEUROCHEMISTRY, WILEY INTERSCIENCE, NEW YORK, NY, US, vol. 98, no. 2, 1 July 2006 (2006-07-01), pages 543 - 554, XP009125244, ISSN: 0022-3042, [retrieved on 20060518] *
KIKUSUI, T. ET AL: "Alarm pheromone enhances stress-induced hyperthermia in rats", PHYSIOLOGY & BEHAVIOUR, vol. 72, 2001, pages 45 - 50, XP002554167 *
KIYOKAWA, Y. ET AL: "Alarm pheromone that aggravates stress-induced hyperthermia is soluble in water", CHEM. SENSES, vol. 30, 2005, pages 513 - 519, XP002554168 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013138948A1 (fr) * 2012-03-19 2013-09-26 Rugen Therapeutics R & D (Shanghai) Co., Ltd. Protéines de détection de calcium sensibles à l'oxydoréduction et leurs méthodes d'utilisation
EP2772487A1 (fr) * 2013-02-27 2014-09-03 Université de Lausanne Phéromones d'alarme et leur utilisation
CN108168967A (zh) * 2017-12-12 2018-06-15 重庆师范大学 一种鱼类预警信息素的制备方法

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