Central and peripheral endocrine correlates of the immobility reaction in the toad Bufo bufo

Behavioural

Processes, 24 (1991) 1-7

0 1991 Elsevier

BEPROC

Science Publishers

B.V. 0376-6357/91/$03.50

00352

Central and peripheral the immobility

di Fisiologia

correlates

of

reaction in the toad Bufo bufo

C. Lupo, lstituto

endocrine

Umana,

L. Lodi, G. Paluffi Universitd

di Siena,

(Accepted

and A. Viti

Via del

2 February

Laterino

8, 53100

Siena,

Italy

1991)

Abstract The

immobility

been

observed

lates

were

which

reaction, in several

established

the

duration

study

was

reaction

and

central

brain

and plasma

active

in the of

present

which animal

sex steroids toads

males

A,

animals ones.

immobility with

higher

Lower

T

whereas

plasma

binding

found

to exist

controlling through

Key

levels

between

behaviour. mechanism.

immobility

the

brain

reaction;

Mating

may

Sex steroid;

less

induced

higher

was

negatively

to

affinity A I\

and

in

than

B. Sexually is

than

active inactive

associated with

rnr;nrnr=l I-cL.lp,“La,

with plasma

off-c-t CllC:cI

peripheral

susceptibility

the

sexually

by inversion

correlated

r3n~,r;t\, LclpJoLlLy.

at central influence

immobility

susceptible

susceptible

and

h;nAinn V”‘““‘6

parameters

was

of the

capacity

to be more

in

bufo,

aim

between

susceptible

were

Bufo The

has corre-

of male and female

found

more

toad

binding

reaction

were

were

(T)

response,

endocrine

mating.

a relation

groups

immobility

of sex steroids in

endocrine

immobility

words:

in four

to the immobility rnrralz,tnrl with ._“IIL.“LL” ““1111 T

an endocrine

of

fear Its

in the

and

Testosterone

Females

males

capacity

sexual activity. Susceptibility EOV ctorn;r(c =“A y”-“““L-‘y nnciti\,ol\, 3cI\ 31L_1”IUJ “II”

the

(B).

and

by sex

possibility

studied

in which

Amphibians.

cristatus

influenced

the

as an innate

including

receptors. were

+pressure

be regarded

Triturus is

investigate hormonal

(A) and by inversion to

newt

immobility

to

and inactive

may species

level,

\a,-,~ ““(13

both

to immobility

Toad

Introduction The

immobility

reaction

has been

species

following

pinching

or clipping

form

of induction

consists

of inversion

observed of the

in several skin,

and restriction.

but

vertebrate the

most

A. wicielv . . ‘__.,

and

invertebrate

frequently

;rcrentdr.-____

studied

Punlanatinn -,.r .-.._..-..

2 is that

it constitutes

and the the

an innate

duration

approach

electric

or

shock

presence

(Gallup

neurophysiological procedure

of

response Carli

of

inversion

is quite

a predator

et al., ‘1970)

aspects

have

and

similar

to predation

is accordingly

clip

for

(Gallup

application

all

the

from

extensively

evoking

1971;

Hennig

conspecifics

studied

(Carli,

different

and Thompson,

by fear

et al.,

and isolation been

(Ratner

prolonged

1977;

induction

in

et

Carli

197h),

1972).

rabbrt

et al.,

methods

like

al.,

(Gallup, the

1960)

stimuli

Its

using

1984)

the

and

(Kumazawa,

the

1963;

et al., 1984).

The

endocrine

rabbit

show

that

sensitization Lupo plasma

mus

induced

is related

to an increase

(Carli was

et al., 1979).

observed

after the

Bufo

Rana

temporaria

(Dabrowska

Menescal-De

Oliveira,

found

between

(Lupo

et al., 1983).

by inversion may

be

1985).

duration

demonstrated

assume

that

found brain

the behavioural

function

testosterone

examine

plasma

presence

et al., 1978).

with

the

pressure,

and

in

immobility

transforming

The

metabolism (Lupo

enzymes

steroids

of et al.,

has also

aim of the present

reaction

to

may modulate

and plasma

immobility

influence

It is reasonable

catesbeiana.

in the brain

was levels

induced

and this

animals

hormonal

Triturus

a correiation

central

in non-mating

on the brain.

newt

and peripheral

that

et

l977),

(Hoffmann

crested

by mating

Rana

the

(Nash

and Sabourin,

at central

Vertebrates,

capacity

correlation

of both

in

hypothala-

in Rana pipiens

by skin

of steroid

directly

binding

(Farabollini

on

levels

lower

plasma

in the

is influenced

et al. (1978)

by acting

their

toads

in

a negative

testosterone

metabolism

demonstrated

we

to be much

as in other

was to compare to

bufo

et al., 1984;

and Bufo paracnemis

paper

induced

pressure

the

by Callard

in Amphibians,

non-breeding

Bufo

skin

1982)

levels,

corticosterone

experiment)

and

(Lefebvre

and testosterone

modifications

was

In the amphibian

been

toad

with

by

which

was

with

studied

laevis

In a previous

immobiiity

In the

mediated

testosterone,

Xenopus

and Manikowski,

immobility

combined

1977),

Farabollini

trial

in the

plasma

1981;

(one

has been

pressure

correlated

duration

and immobility

reaction

1988).

in which

carnifex

treatment

in testosterone

induction

(Ternes,

marinus

and

cristatus

increase

both

et al.,

immobility

or skin

in corticosterone

is directly

In acute

immobility

by inversion

(Farabollini

between

a significant

In Amphibians al., 1970),

in serotonin

and susceptibility

observed

moreover was

of immobility

et al., 1980)

levels

levels;

correlates habituation

to a decrease

di Prisco

correlation

and

fear response

of immobility

study

hormone in

levels

breeding

and

sexes.

Methods Male and

and female

35 Y ) and

were

243 + 50 gm (sexually

females

=

inactive The

males and

procedure

i), 91 + 18 =

immobility

$ 6

time was

the

performed of 180

were

bufo

successive active

studied

periods

females

gm (sexually procedure animal

on

repeatedly s. The

considered

as the proper

Bufo

during

(38 $

=

? 0

active

and

the

breeding

12 9 ). Mean

a), 267 + 60

males

=

$ $

season body

gm (sexually

a), 81 f

(35 6

weights inactive

19 gm

(sexually

i).

induction

holding

was

a total

required

0 ?

immobility

placing ing

toads

in different

criterion

of inversion

its

until

back,

according

interval

as the

consisting

to the

between

latency

for assessing

length. the

the At

fully

(Immobility relaxed.

susceptibility first least

immobility

inversion 10

s of

A) involves The

induction

and not

exceed-

and

start

the

immobility

attainment.

Latency

of

were time

3 exceeding

180 s was taken

inversion skin

plus

skin

in addition

were

chloride

glycol

: ethylene

Farner

method

determined

glycol

(6: 1.5 The

Radioimmunoassay

of T and DHT 46 Ci/mmol

levels

activities:

were

determined

the

dried

antiserum

with

coefficient of

the

using

assay

a very

was

IO

antibody-bound mixture. from

51.6

after

5-7%

pg for

T

and

was

H3-steroids

with

and the DHT

were

other

in

purchased

from

New

ether

plasma without

was performed

(<

and

0.1%).

The

10%.

with

England

on

specific

intra-assay

The

separation

experiments

tracers

(E)

Ci/mmol)

E. The

and

50-70%.

tritiated

diethyl

coefficient

4 pg for all

83

steroids

inter-assay

and

performed

activity

was

Estradiol

with

with

: propylene

Wingfield

using

Radioimmunoassay

(specific

cross-reaction

was

the

DHT).

extraction

85-92%).

tracer

for

extraction

standard

on the eluates

by

and dihydro-

in celite to

by

to the

determined (T)

after

internal

Ci/mmol

sample

sample

according

of the

applied

were

Testosterone

plasma

performed

T;

(recovery

low

steroid

The

was

tritiated

of variation

recovery

for

in a single

separation

extract

of

was

chromatography

microcolumns,

range

(specific

chromatographic

in a single

In immobility

clip

sex hormones

session.

by partition

: 1.5)

wooden

Plasma

the immobility

and separation

(1975).

of unsusceptibility.

6) a small

procedure.

the day after

(DHT)

methylene

a condition

(Immobility

to the inversion

radioimmunoassay testosterone

to indicate

pressure

sensitivity of free

and

a charcoal-dextran

Nuclear,

the

antisera

Radim.

Testosterone cytosol

and

18 0 9

(12

containing

a and

6

mM

of the 800

glycerol,

The

extract

nuclei

were

10 nM)

K,PO,,

mM

buffer

incubated

or without

Specific

binding

was

series.

These

bound

: free ratio,

(capacity) dissociation

obtained

differences

were

(Kd)

from

the

diagrams

as a measure

was

as the

of affinity

The

data were and

analyzed

Cochran,

by ANOVA 1980).

and the

Correlations

The

least

significant

performed

0.1 to

tritiated

sites The

between

concentration.

difference by the

’ 1,

of the

(Bmax).

as the ratio nM

T\

of the two binding

intercept

7.4, and

specific

as a function

saturation

abscissa

at pH (from

the

7.4

NaN,,

cytosol (non

the radioactivity

was calculated

were

both with

abscissa and ordinate intercepts and corrected for volume to rl,t,,-;,,rl h.. +l-.,. .W.,.th,.A “I ,.c L”““ly I -..,r., cc -* cl,. aI (17Jl,. /-3ne3\ IDrn+~;-r I”IC1113 \.,,,..,. Y”tT,cz“CLCsIIIII1IC” “y LIlC llIC’1II”” (Snedecor

3 mM

amounts

pg of protein

buffer

at pH

gelatin

times

mM

12

with

sucrose

analysis

/KM \-‘“”

a buffer

by centrifuga-

EDTA,

Ci/mmol)

(1949).

in and

washed

mM

increasing

130

between

in fmol/lOO

i) and

obtained

1 mM

toctnctnrnnn .._.,I.,JLLI”IIL

to Scatchard

both

a and 11

and 2 mg/ml

with

as the difference

according

calculated

constant

raciininort SW V.VI., _,L

incubating

glycerol

and 250

the saturation

activity

by (21

homogenized was

K,PO,,

thioglycerol

$

10%

pellet

NaN,

of test tubes

are reported

plotted

was NaN,,

20 mM

brain

in 328

nuclei

3 mM

(specific

hindinob ..,,.. Y”‘b,

the

g. Cytosol

g.The

12 mM

series

mM

at 800

at IOOCOO g. For

in two (total

subject 3

MgCI,,

of 1,2,6,7-H’-testosterone

binding)

each

EDTA,

containing

NaMoO,,

mM

centrifuged

in

encephalon

at IO0000

5 mM

with

100 was

from

1

g supernatant

extracted

10%

Tissue

determined

whole

7.4 and centrifuged

10 mM

and was

i).

was

from

KzPO,,

at pH

containing

capacity

obtained

20

thioglycerol tion

binding nuclei

(LSD)

Spearman

test rank

test.

Results The were

immobility more

and latency

susceptible

than

durations females

(means to

and SE) are shown

immobility

B (ANOVA

in Table

F = 2.37,

1. Males

df = 3/116,

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h

behavioural bufo

function that

suggest

hormones

to their

depends

upon

brain

behaviour, ments

that

but

a lower

acting

incorporate features

the

brain.

encephalon a higher

capacity,

may

the

toad

reaction

hormones

of testosterone higher

at central

binding

values

Bufo

of sexual

immobility

sexual

to the reflect

both

laboratory

and

upon

affinity

others

and Pough,

in the binding

in the

of circulating

in the brain.

of anurans field

and that

owing

conversion

findings

by a specific

and therefore

independently,

(Taigen

Our

is controlled

in the cycle

characteristics

some

interrelated

sites

or to increased

endocrine

on

In fact we found

toads

hormones

The

directly behaviour

reproductive

level.

of mating

sexual

receptor

the

and peripheral

by acting

reproductive

many

in concert. data

aspects

Carefully are

of ecology designed

required

to

and

experi-

assess

these

1985).

References Barker Jorgensen,

C., 1974.

the Amphibian, Callard,

C.V.,

tissues

Petro,

of the

G., 1977.

Carli,

G., Farabollini,

Carli,

G.,

Z.

hypnosis

Dabrowska,

F. and

Behav.

Farabollini, Farabollini,

Physiology

brain

and non-neural

in the

Endocr., Rec.,

F., 1984. C., 1979.

S., 1982.

(Editor),

of

34: 18-25.

27. 123-143.

Physiological

characteristics

Behav.

Res.,

Plasma

Neurosct.

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