Modulations of olfactory responses by GAP junctions uncoupling in the OM.

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Modulations of olfactory responses by GAP junctions uncoupling in the OM.

Mikael Le Bourhis, Bertrand Bryche, Denise Grebert, Edith Pajot, Patrice Congar, Nicolas Meunier

To cite this version:

Mikael Le Bourhis, Bertrand Bryche, Denise Grebert, Edith Pajot, Patrice Congar, et al.. Modula- tions of olfactory responses by GAP junctions uncoupling in the OM.. XXII. Congress of the European Chemoreception Research Organization (ECRO 2013), Aug 2013, Louvain, Belgium. Chemical Senses, 39, 2013, Abstracts from the XXIInd Congress of the European Chemoreception Research Organiza- tion, ECRO 2013. �10.1093/chemse/bjt055�. �hal-01349543�

Mikael Le Bourhis, PhD student mlebourhis@jouy.inra.fr 06.50.98.50.42 / 01.34.65.25.55

INRA, centre de Jouy-en-Josas www.inra.fr Neurobiologie de l'Olfaction et Modélisation en Imagerie (NOeMI) Biologie de l'Olfaction et Biosenseurs (B.O.B.) Bât 440 Domaine de Vilvert 78352 Jouy en Josas cedex France

Glial cells not only act as supporting cells, they are clearly involved in nervous communication. Modulation of odorant detection takes place as early as the peripheral level, on the olfactory sensory neurons. Those neurons are surrounded by glial like cells called sustentacular cells (SCs). These cells maintain both the structural and ionic integrity of the olfactory epithelium. They are electrically coupled through GAP junctions (Vogalis et al., 2005).

Using endothelin (ET) and carbenoxolone (CBX) to uncouple GAP junctions (Blomstread et al., 2004), the aim of this study is to test the involvement of SC’s GAP junctions in olfactory responses.

Cartilage

OM SCs

Δ F/F

98,0 100,0 102,0 104,0 106,0 108,0 110,0

0 50 100 150 200 250 300

ET ¹⁰

^M

n= 52

Time (s)

ET ¹⁰

^M

Δ F/F

98,0 100,0 104,0 108,0 112,0 116,0 118,0

0 50 100 150 200 250

Time (s)

n= 6

I - ET activates SCs ex vivo Method : Olfactory Mucosa (OM) slices 200µm (Wistar P0-P4), loaded with Oregon Green (488nm), Calcium Imaging.

Tissue response Single cell response

I - ET evokes both tonic and phasic calcium responses in the

SCs.

SCs respond in vitro (Gouadon et al., 2010) and ex vivo to ET. This peptide acts as an autocrine and paracrine factor. ET induces uncoupling of SC’s GAP junctions both in vitro and ex vivo, like the classically used GAP junction uncoupling agent CBX.

Such modulations affect olfactory responses. Both ET and CBX treatments increase EOG signal repolarization kinetics. Our results are consistent with an implication of glial-like cells in the peripheral modulation of the olfactory information.

This has to be confirmed using patch clamp on the knobs of olfactory sensory neurons.

 Blomstrand F et al., Endothelins regulate astrocyte gap junctions in rat hippocampal slices. Eur J Neurosci. (2004)

 Gouadon et al., Endothelin evokes distinct calcium transients in neuronal and non-neuronal cells of rat olfactory mucosa primary cultures. Neuroscience. (2010)

 Fivos Vogalis et al., Electrical Coupling in Sustentacular Cells of the Mouse Olfactory Epithelium. J. Neurophysiol. (2005)

III - ET and CBX modify olfactory responses.

Method : Hemi-Head (P30), Electro-OlfactoGram (EOG), Isoamyl Acetate 1:10 in mineral oil, n = 6 in each group.

III - GAP

junctions of SCs are involved in

olfactory responses.

Repolarization kinetics of the olfactory response , but not amplitude, are affected by GAP junctions uncoupling.

II

II’ III IV II

II’ III IV I

MO Drugs

Saline

BO Channel 1

Channel 2 _{0 10}

Time (s)

Voltage (mV)

-20 -10 0

IA 10^-1 1:10 (200 ms)

+ ET / CBX

Decay slope Amplitude

**

Normalized Amplitude (% of control)

0 20 40 60 80 100 120

Normalized Decay Slope (% of control)

0 20 40 60 80 100 120

Saline

CBX 10

M

CBX

**

Animals were pretreated by 3 intranasal injections before recordings : -9h ; -6h ; -3h.

vehicle or antagonists of ET receptors (BQs).

ET

Saline ET 10

M

Vehicle BQs

0 20 40 60 80 100 120

Normalized Amplitude (% of control)

0 20 40 60 80 100 120

Normalized Decay Slope (% of control)

0 20 40 60 80 100 120

Normalized Amplitude (% of control)

0 20 40 60 80 100 120

Normalized Decay Slope (% of control) Treatment

(Vehicle Drugs )

Basal EOG

recording Post-treatment EOG recordings

-2’ 0 2’ 4’ 6’

Control EOG

recording Post-treatment EOG recordings Treatment

(Vehicle ± Drugs)

II - Plasticity of GAP junctions ex vivo and in vitro.

Method : Olfactory Mucosa explant (Wistar P7), loaded with Oregon Green, Electric stimulation and Calcium Imaging.

n > 20 for each group.

II - ET and CBX uncouple GAP junctions of SCs.

Method : Olfactory Mucosa Primary Culture,

Whole cell Voltage Clamp. n > 15 for each group.

CBX and ET ​​decrease spreading of the calcium wave.

% ΔF/F

0 20 40 60 80 100 120

ROI 1 ROI 2 ROI 3 ROI 4 ROI 5 ROI 6 ROI 7

ATP 10

M Saline

CBX 10

M ET 10

M

***

*** ***

###

###

###

CBX and ET ​​increase membrane resistance of SCs.

ET 10^-7M CBX 10^-4M ATP10^-7M Saline

0 50 100 150 200 250

Basal

Drugs ***

**

Normalized membrane resistance

** : P<0,01

*** : P<0,001