Functional Asymmetry in the Olfactory System of a Flatfish, the

Senegalese Sole (Solea senegalensis)

Zélia Velez1,2,3, Peter C. Hubbard1, Kevin J. Weham3, Eduardo N. Barata1,2, Jörg D. Hardege3

and Adelino V.M. Canário1

1. Centro de Ciências do Mar, Faro, Portugal; 2. Departamento de Biologia,

Universidade de Évora, Portugal; 3. University of Hull, UK

High commercial value – increased aquaculture effort.Flatfish; left (lower) nostril does not migrate.

Functional asymmetry in the olfactory system?Lower epithelium – prey location/identificationUpper epithelium – chemical communication

The Selenagese Sole (Solea senegalensis):

Methods:

Electro-olfactogram (EOG) recorded from the olfactory epithelium:

Olfactory potency – identification of odorantsCross-adaptation – receptor specificityPharmacological inhibition - transduction pathways

Chromatography of biological fluids:

Solid-phase extraction – crude separation of odorantsHPLC – fine separation and purification of odorantsLC-MS – identification of odorants

Methods:

The ragworm Hediste diversicolor is one of the sole’s main prey.They live buried in the sand or mud (of estuaries).Bile acids are potent odorants in fish.

Food- and conspecific-related odorants

Bile and intestinal fluidRagworm-conditioned water

C-18 Sep-Pak

Total; EOG

Filtrate HPLCand EOG

EluateEOG

Methods:

EOG is a spacio-temporal summation of generator potentials of olfactory receptor neurones. L-cysteine is detected with equal sensitivity by the two olfactory epithelia; responses are normalised to that of 10-3 M L-cysteine.

The electro-olfactogram (EOG)

Velez et al. (2005) Physiol. Biochem. Zool. 78: 756-765

Results I:

Fractions 1 and 2 contain the odorants of interest; active odorant identified using LC-MS

Fraction

Total 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Nor

mal

ised

EO

G A

mpl

itude

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Upper olfactory epitheliumLower olfactory epithelium

**

*

**

HPLC fractionation of ragworm-conditioned water

Results I:

Active odorant identified as 1-methyl-L-tryptophan. Ragworms release this amino acid, along with L-

phenylalanine and L-glutamine

1-methyl-L-tryptophan

Identification of ragworm-related odorants:

Results I:

Lower epithelium has greater sensitivity to 1-methyl-L-tryptophan.

Ragworm odorant (10-3 M)

% c

ontr

ol r

espo

nse

0

10

20

30

40

50 Upper olfactory epitheliumLower olfactory epithelium

**

Log [ragworm odorant] (M)

-6 -5 -4 -3

Nor

mal

ised

EO

G A

mpl

itude

0.0

0.2

0.4

0.6

0.8Upper olfactory epitheliumLower olfactory epithelium

*** Elevation** Slope

Olfactory sensitivityCross-adaptation - selectivity

Results I:

Log(Dilution)

-6.0 -5.0 -4.0 -3.0

No

rma

lised

EO

G A

mp

litud

e

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8Upper nostril Lower nostril

B

***Elevation***Slope

Log(Dilution)

-6.0 -5.0 -4.0 -3.0

Nor

mal

ised

EO

G A

mpl

itude

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6Upper nostril Lower nostril

A

***Elevation**Slope

Log(Dilution)

-5.0 -4.0 -3.0 -2.0

Nor

mal

ised

EO

G A

mpl

itude

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4Upper nostril Lower nostril

C

*** Elevation** Slope

Bile Intestinal fluid Mucus

Conspecific body fluids are consistently better detected by the upper olfactory epithelium

Olfactory sensitivity to conspecific odorants

Velez et al. (2007) Gen. Comp. Endocrinol. 153: 418-425

Results I:

HPLC fraction

Total 1 2 3 4 5 6 7 8 9 10

No

rma

lise

d E

OG

am

plit

ud

e

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8upper epitheliumlower epithelium*

*

HPLC fraction

Total 1 2 3 4 5 6 7 8 9 10B

ile a

cid

conc

entr

atio

n (m

g.m

l-1)0

1

2

3

4

5

6

7

Bile acids contribute about 40% of the olfactory potency of intestinal fluid.

Bile acids in fraction 4 were identified by LC-MS.

Chromatographic separation of odorantsin intestinal fluid

Velez et al. (2009) J. Comp. Physiol. A 195: 691-698

Results I:

Two major bile acids identified in the intestinal fluid (and bile fluid).

A third, minor, bile acid was not identified.

Identification of odorants in intestinal fluid:

Velez et al. (2009) J. Comp. Physiol. A 195: 691-698

Taurocholic acid Taurolithocholic acid

Results I:

(a)

log [TCH] (M)

-9 -8 -7 -6 -5

No

rma

lise

d E

OG

am

plit

ud

e

0.0

0.2

0.4

0.6

0.8

1.0upper epitheliumlower epithelium

**Elevation**Slope

(b)

log [TLC] (M)

-9 -8 -7 -6 -5

Nor

mal

ised

EO

G a

mpl

itude

0.0

0.2

0.4

0.6

0.8

1.0upper epitheliumlower epithelium

(a)

log [TCH] (M)

-9 -8 -7 -6 -5

No

rma

lise

d E

OG

am

plit

ud

e

0.0

0.2

0.4

0.6

0.8

1.0upper epitheliumlower epithelium

**Elevation**Slope

(b)

log [TLC] (M)

-9 -8 -7 -6 -5

Nor

mal

ised

EO

G a

mpl

itude

0.0

0.2

0.4

0.6

0.8

1.0upper epitheliumlower epithelium

Taurocholic acid Taurolithocholic acid

Sole release taurocholic acid – this is detected better by the upper olfactory epithelium

Olfactory sensitivity to bile acids

Conclusions I:

Prey-related odorants – including 1-methyl-L-tryptophan – are better detected by the lower olfactory epithelium.

Conspecific-related odorants – including taurocholic acid – are better detected by the upper olfactory epithelium.

There is functional asymmetry in the olfactory system of this flatfish.

Are the receptors and transduction pathways different?

Results II: Cross-adaptation

Lower epithelium has greater specificity for the ragworm odorant 1-methyl-L-tryptophan.

Adapting Solution (10-3 M)

L-cysteine Glycine L-phenylalanine

% c

ontr

ol r

espo

nse

0

20

40

60

80

100 Upper olfactory epithelium Lower olfactory epithelium

**

**

**

1-methyl-L-tryptophan

Results II: Cross-adaptation

Upper epithelium has greater specificity for taurocholic acid.

Adapting solution

L-cysteine 1-M-L-T Cholic acid

% c

ontr

ol r

espo

nse

0

20

40

60

80

100 UpperLower

*** ***

***

taurocholic acid

Results II: Transduction

L-cysteine is detected equally by the two epitheliaThe effects of the two drugs are the same in the two epithliaThe transduction pathway for L-cysteine is mainly via PLC

U73122 – PLC inhibitorSQ-22536 – AC inhibitor

U73122 SQ-22536 Both

% c

ontr

ol r

espo

nse

0

20

40

60

80

100 Upper

Lower

a x

b y

c x

log[U73122] (M)

-9 -8 -7 -6

Nor

ma

lise

d E

OG

Am

plitu

de

0.0

0.2

0.4

0.6

0.8

1.0

L-cysteine L-cysteine

Results II: Transduction

The transduction pathway for 1-methyl-L-tryptophan is mainly PLC mediated

There are differences between the two epithelia

U73122 SQ-22536 Both

% c

ontr

ol r

espo

nse

0

20

40

60

80

100

120 UpperLower

**

**

a

b

cx

y

x

1-methyl-L-tryptophan

U73122 – PLCSQ-22536 – AC

Results II: Transduction

Both phospholipase C and adenylate cyclase are involved in transduction of taurocholic acid

There are differences in the two epithelia

U73122 SQ-22536 Both

% c

ontr

ol r

espo

nse

0

20

40

60

80

100 UpperLower

***

**

*

a

abb

x

y

x

taurocholic acid

U73122 – PLCSQ-22536 – AC

Conclusions II:

There are specific receptors for 1-methyl-L-tryptophan in the lower epithelium.

There are specific receptors for taurocholic acid in the upper epithelium.

The transduction pathway for 1-methyl-L-tryptophan is mainly phospholipase C mediated.

The transduction pathway(s) for taurocholic acid is/are mediated by both phospholipase C and adenylate cyclase.

Summary:

Specific receptors in the lower olfacotry epithelium for prey-related odorants (1-methyl-L-tryptophan)

Specific receptors in the upper olfactory epithelium for conspecific-related odorants (taurocholic acid)

This suggests specialisation of the two epithelia – functional asymmetry

Does the processing of the olfactory information in the olfactory bulb (and beyond) also show asymmetry?

The End:

Thank you for your attention

Funded by FCT grants SFRH/BD/16242/2004 and POCI/BIA-BMC/55467/2004