Hypertonicity-induced

cation

channels

rescue

cells

from

staurosporine-elicited

apoptosis

Cell

shrinkage

is

one

of

the

earliest

events

during

apoptosis

Cell

shrinkage

also

occurs

upon

hypertonic

stress

and

previous

work

has

shown

that

hypertonicity-induced

cation

channels

-LRB-

HICCs

-RRB-

underlie

a

highly

efficient

mechanism

of

recovery

from

cell

shrinkage

called

the

regulatory

volume

increase

-LRB-

RVI

-RRB-

in

many

cell

types

Here

the

effects

of

HICC

activation

on

staurosporine-induced

apoptotic

volume

decrease

-LRB-

AVD

-RRB-

and

apoptosis

were

studied

in

HeLa

cells

by

means

of

electronic

cell

sizing

and

whole-cell

patch-clamp

recording

It

was

found

that

hypertonic

stress

reduces

staurosporine-induced

AVD

and

cell

death

-LRB-

associated

with

activation

and

DNA

fragmentation

-RRB-

and

that

this

effect

was

actually

due

to

activation

of

the

HICC

On

the

other

hand

staurosporine

was

found

to

significantly

reduce

osmotic

HICC

activation

It

is

concluded

that

AVD

and

RVI

reflect

two

fundamentally

distinct

functional

modes

in

terms

of

the

activity

and

role

of

the

HICC

in

a

shrunken

cell

Our

results

also

demonstrate

for

the

first

time

the

ability

of

the

HICC

to

rescue

cells

from

the

process

of

programmed

cell

death

Introduction

It

has

become

increasingly

evident

in

recent

years

that

the

mechanisms

of

cell

volume

regulation

are

employed

in

a

variety

of

physiological

processes

in

addition

to

just

the

maintenance

of

cell

homeostasis

Among

these

processes

are

the

coordination

of

transport

across

the

apical

and

basolateral

membranes

in

epithelia

the

locomotion

of

cells

the

regulation

of

metabolic

processes

in

the

liver

and

most

notably

the

control

of

cell

proliferation

and

apoptosis

-LSB-

-RSB-

Since

the

rates

of

ion

transport

through

channels

are

some

orders

of

magnitude

higher

than

those

achieved

by

transporters

-LRB-

carriers

and

pumps

-RRB-

any

modulation

of

ion

channel

activity

may

serve

as

a

rapid

and

efficient

mechanism

of

osmolyte

transport

and

cell

volume

regulation

Accordingly

hypertonicity-induced

cation

channels

-LRB-

HICCs

-RRB-

were

found

to

be

the

main

mediators

of

the

regulatory

volume

increase

-LRB-

RVI

-RRB-

of

a

shrunken

cell

whenever

a

system

was

analysed

in

a

quantitative

fashion

-LSB-

-RSB-

Despite

the

low

selectivity

of

HICCs

for

over

K

activation

of

these

channels

leads

to

an

actual

net

uptake

of

cations

which

is

mainly

due

to

the

membrane

voltage

which

is

negative

intracellularly

Anions

and

osmotically

obliged

water

follow

allowing

the

cell

to

complete

the

process

of

RVI

-LSB-

-RSB-

Persistent

isotonic

shrinkage

of

a

given

cell

as

opposed

to

hypertonicity-induced

shrinkage

is

one

of

the

hallmarks

of

apoptosis

In

many

instances

this

apoptotic

volume

decrease

-LRB-

AVD

-RRB-

employs

the

activation

of

K

channels

which

serve

as

the

main

routes

of

K

exit

-LSB-

-RSB-

Cl

channels

also

should

open

with

this

parallel

transport

strongly

depending

however

on

whether

or

not

the

electro-chemical

driving

forces

actually

favour

conductive

Cl

export

-LSB-

-RSB-

The

question

is

thus

raised

of

how

hypertonic

stress

might

interfere

with

AVD

Would

osmotic

shrinkage

facilitate

the

process

Or

would

the

activation

of

HICCs

or

RVI

oppose

the

induction

of

apoptosis

Materials

and

methods

Cell

culture

Human

cervix

HeLa

cells

were

grown

as

monolayers

in

minimum

essential

medium

supplemented

with

fetal

bovine

serum

μg/ml

streptomycin

and

IU/ml

penicillin

under

at

°C

For

the

experiments

cells

were

mechanically

detached

from

the

plastic

substrate

of

the

culture

flasks

with

a

jet

of

culture

medium

and

then

kept

in

suspension

for

min

Determination

of

cell

volume

Changes

of

HeLa

cell

volume

were

quantified

by

means

of

electronic

cell

sizing

on

a

Coulter-type

analyzer

-LRB-

Sysmex

Kobe

Japan

-RRB-

as

previously

described

-LSB-

-RSB-

The

experimental

solution

contained

-LRB-

in

mM

-RRB-

NaCl

KCl

HEPES

Osmolality

was

adjusted

to

values

from

to

mosmol/kg-H

by

adding

mannitol

under

osmometric

control

-LRB-

Vogel

Giessen

Germany

-RRB-

Measurements

were

performed

at

room

temperature

Patch-clamp

experiments

Membrane

currents

were

recorded

in

the

fast

whole-cell

mode

of

the

patch-clamp

technique

using

MΩ

borosilicate

pipettes

Currents

were

recorded

with

an

Axopatch

amplifier

-LRB-

Molecular

Devices

Union

City

CA

-RRB-

filtered

at

kHz

with

a

four-pole

Bessel

filter

and

digitized

at

kHz

pClamp

software

-LRB-

Molecular

Devices

-RRB-

was

used

for

control

of

the

pulse

protocol

as

well

as

for

data

acquisition

and

analysis

Series

resistance

was

MΩ

and

was

compensated

-LRB-

by

-RRB-

to

minimize

voltage

errors

In

the

experiments

voltage

ramps

from

to

mV

and

of

s

duration

were

applied

every

s

holding

voltage

was

mV

The

bath

solution

-LRB-

pH

-RRB-

contained

-LRB-

in

mM

-RRB-

NaCl

Na-gluconate

TEA-Cl

HEPES

d-glucose

Osmolality

was

adjusted

to

-LRB-

isotonic

-RRB-

and

mosmol/kg-H

-LRB-

hypertonic

-RRB-

by

the

addition

of

mannitol

The

pipette

solution

-LRB-

pH

mosmol/kg-H

-RRB-

contained

-LRB-

in

mmol/l

-RRB-

NaCl

Na-gluconate

TEA-Cl

HEPES

EGTA

With

these

ion

gradients

the

equilibrium

potentials

for

and

Cl

-LRB-

as

the

only

permeant

ions

-RRB-

are

set

at

and

mV

respectively

-LSB-

-RSB-

Experiments

were

performed

at

room

temperature

Monitoring

apoptotic

cell

death

Cell

viability

and

activity

were

determined

by

use

of

a

calorimetric

MTT

assay

-LRB-

Cell

Counting

Dojindo

Kumamoto

Japan

-RRB-

and

a

fluorometric

probe

-LRB-

Apo-ONE

Homogeneous

Assay

Promega

Madison

WI

-RRB-

respectively

Fragmentation

of

DNA

was

determined

by

means

of

a

photometric

enzyme

immunoassay

-LRB-

Cell

Death

Detection

ELISAPLUS

Roche

Applied

Science

Diagnostics

Mannheim

Germany

-RRB-

with

HeLa

cells

plated

on

plates

Following

exposure

to

the

various

experimental

conditions

cells

were

lysed

and

centrifuged

at

The

cytoplasmic

-LRB-

DNA

containing

-RRB-

fraction

was

then

transferred

to

streptavidin-coated

microtiter

plates

that

had

been

incubated

with

a

biotinylated

monoclonal

anti-histone

antibody

Finally

the

amount

of

the

fragmented

DNA

on

bound

nucleosomes

was

determined

with

a

peroxidase-conjugated

monoclonal

anti-DNA

antibody

and

the

substrate

ABTS

-LSB-

azino-di

-LRB-

sulfonate

-RRB-

-RSB-

which

was

quantified

photometrically

at

nm

In

all

measurements

cells

were

incubated

for

h

at

°C

Testing

for

necrosis

and

late

apoptosis

Necrosis

and

late

apoptosis

of

cells

were

quantified

fluorometrically

on

the

basis

of

a

propidium

iodide

staining

of

nuclei

-LRB-

excitation/emission

at

nm

-RRB-

performed

with

reference

to

an

overall

staining

of

cells

by

Hoechst

-LRB-

excitation/emission

at

nm

-RRB-

Chemicals

All

reagents

were

purchased

from

Sigma

Aldrich

-LRB-

Tokyo

Japan

-RRB-

Statistical

analysis

Data

are

presented

as

mean

values

SEM

with

n

denoting

the

number

of

observations

Student

's

t

tests

for

paired

and

unpaired

data

were

used

as

appropriate

and

P

was

considered

significant

Results

and

discussion

As

was

determined

by

means

of

electronic

cell

sizing

increasing

osmolality

from

mosmol/kg-H

to

and

mosmol/kg-H

led

to

an

initial

shrinkage

of

HeLa

cells

to

and

of

the

control

level

respectively

-LRB-

n

Fig.

-RRB-

When

plotted

as

a

function

of

-LRB-

which

is

equivalent

to

a

Boyle-van

t

Hoff

graph

-LSB-

-RSB-

-RRB-

these

data

could

readily

be

fitted

by

a

straight

line

its

intercept

with

the

y-axis

yields

the

osmotically

active

versus

inactive

space

of

the

cells

with

the

latter

amounting

to

of

total

cell

volume

-LRB-

Fig.

Insert

-RRB-

This

osmometric

behaviour

of

HeLa

cells

was

then

followed

by

an

active

volume

recovery

to

and

in

min

of

hypertonic

stress

-LRB-

Fig.

b

-RRB-

The

process

of

regulatory

volume

increase

-LRB-

RVI

-RRB-

occurred

at

essentially

constant

rates

that

equalled

and

min

respectively

in

the

range

of

min

after

application

of

hypertonicity

Fig.

HeLa

cell

shrinkage

and

RVI

determined

by

electronic

cell

sizing

-LRB-

a

-RRB-

The

volume

response

of

cells

to

increases

in

osmolality

-LRB-

from

mosmol/kg-H

-RRB-

to

and

mosmol/kg-H

at

time

zero

-LRB-

arrow

-RRB-

are

depicted

The

insert

gives

the

initial

-LRB-

passive

-RRB-

amount

of

cell

shrinkage

as

a

function

of

-LRB-

equivalent

to

a

Boyle-van

t

Hoff

plot

of

these

data

see

text

for

details

-RRB-

osmol

-LRB-

osmol/kg-H

-RRB-

is

the

value

under

isotonic

conditions

-LRB-

b

-RRB-

The

volume

of

HeLa

cells

at

min

of

various

levels

of

hypertonic

stress

after

partial

volume

recovery

-LRB-

RVI

-RRB-

had

been

achieved

n

for

each

experimental

condition

It

has

become

increasingly

evident

in

recent

years

that

persistent

cell

shrinkage

is

one

of

the

hallmarks

of

apoptosis

-LSB-

-RSB-

Also

it

has

recently

been

found

that

after

stimulation

with

staurosporine

-LRB-

STS

-RRB-

TNFα

or

Fas

ligand

HeLa

cells

lose

their

ability

to

undergo

RVI

-LRB-

-RRB-

To

test

whether

HeLa

cell

shrinkage

and/or

RVI

interferes

with

apoptotic

volume

decrease

-LRB-

AVD

-RRB-

μM

STS

was

used

to

induce

apoptosis

in

HeLa

cells

-LSB-

-RSB-

and

a

series

of

volume

measurements

was

made

This

was

done

under

isotonic

conditions

-LRB-

mosmol/kg-H

-RRB-

and

under

hypertonic

conditions

in

which

osmolality

was

increased

to

and

mosmol/kg-H

-LRB-

n

-RRB-

Within

an

experimental

time

frame

of

min

STS

was

found

to

reduce

cell

volume

to

that

of

the

control

under

isotonic

conditions

-LRB-

Fig.

diamonds

-RRB-

min

after

osmolality

was

increased

to

and

mosmol/kg-H

cell

volume

equalled

and

of

control

respectively

These

values

are

virtually

identical

to

the

values

of

purely

passive

or

physical

responses

to

hypertonic

stimulation

in

the

absence

of

STS

-LRB-

cf.

to

Fig.

-RRB-

Moreover

an

osmotically

inactive

space

of

could

be

calculated

from

these

measurements

this

value

is

very

similar

to

that

obtained

in

the

control

experiments

-LRB-

see

above

-RRB-

Of

note

no

RVI

followed

hypertonicity-induced

cell

shrinkage

when

STS

was

present

This

indicates

that

the

RVI

response

was

completely

inhibited

by

STS

-LRB-

or

surpassed

by

the

STS-induced

AVD

process

-RRB-

a

phenomenon

which

clearly

holds

for

the

full

h

period

after

STS

application

Using

the

respective

values

for

volume

at

min

as

passive

reference

values

AVD

was

computed

to

be

and

for

and

mosmol/kg-H

respectively

-LRB-

calculated

as

the

volume

at

min

minus

the

volume

at

min

-RRB-

So

as

summarized

in

Fig.

a

decrease

of

AVD

was

observed

with

increasing

hypertonic

stress

On

the

one

hand

then

hypertonic

stress

appears

to

suppress

STS-induced

AVD

on

the

other

hand

STS

seems

to

abolish

hypertonicity-induced

RVI

Also

of

note

in

this

respect

is

a

recent

study

on

HeLa

cells

in

which

tyrosine

kinases

PKC

and

MAPK

were

identified

as

part

of

the

signalling

machinery

employed

in

the

activation

of

HICC

and

RVI

-LSB-

-RSB-

Given

the

non-specificity

of

STS

with

respect

to

its

inhibition

of

various

protein

kinases

it

is

not

surprising

that

RVI

is

inhibited

in

the

presence

of

the

compound

while

AVD

is

induced

Fig.

of

hypertonic

stress

on

apoptotic

volume

decrease

-LRB-

AVD

-RRB-

-LRB-

a

-RRB-

At

time

zero

-LRB-

arrow

-RRB-

μM

staurosporine

-LRB-

STS

-RRB-

was

added

to

induce

apoptotic

shrinkage

of

HeLa

cells

under

isotonic

conditions

-LRB-

-RRB-

or

together

with

an

increase

of

osmolality

to

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

mosmol/kg-H

Hypertonic

cell

shrinkage

at

min

was

very

similar

in

the

presence

and

absence

-LRB-

see

Fig.

-RRB-

of

STS

-LRB-

b

-RRB-

AVD

under

isotonic

conditions

-LRB-

volume

at

min

minus

volume

at

min

-RRB-

and

hypertonic

conditions

-LRB-

volume

at

min

minus

volume

at

min

-RRB-

n

for

each

experimental

condition

To

study

the

interference

of

STS-induced

AVD

with

hypertonicity-induced

RVI

in

more

detail

experiments

were

performed

in

which

osmolality

was

increased

from

to

mosmol/kg-H

at

time

zero

-LRB-

see

Fig.

arrow

-RRB-

In

addition

μM

STS

was

applied

either

h

before

-LRB-

t

min

-RRB-

h

before

-LRB-

t

min

-RRB-

at

the

same

time

as

-LRB-

t

-RRB-

or

h

after

-LRB-

t

min

-RRB-

hypertonic

stimulation

-LRB-

see

Fig.

arrowheads

-RRB-

AVD

was

then

computed

from

the

overall

changes

in

cell

volume

-LRB-

i.e.

the

volume

at

or

min

respectively

minus

the

volume

at

min

-RRB-

minus

the

amount

of

passive

cell

shrinkage

-LRB-

i.e.

the

volume

at

min

minus

the

volume

at

min

-RRB-

Hypertonic

stress

-LRB-

mosmol/kg-H

-RRB-

significantly

reduced

AVD

from

the

control

value

of

-LRB-

Fig.

also

marked

as

STS-ISO

in

Fig.

-RRB-

to

or

-LRB-

Fig.

STS-HYPER

or

-RRB-

when

given

simultaneously

with

or

h

before

application

of

STS

-LRB-

n

P

each

-RRB-

respectively

Also

apparent

from

these

experiments

is

that

no

RVI

occurred

in

the

presence

of

STS

It

is

noteworthy

that

increasing

the

osmolality

to

mosmol/kg-H

after

pretreatment

with

STS

still

led

to

a

markedly

reduced

AVD

of

-LRB-

P

Fig.

-RRB-

A

hypertonic

stimulation

after

pretreatment

with

STS

however

was

not

effective

at

suppressing

AVD

-LRB-

Fig.

-RRB-

These

results

suggest

that

hypertonic

stimulation

of

HeLa

cells

is

somehow

interfering

with

the

process

of

AVD

when

applied

in

the

time

range

of

h

before

to

h

after

the

application

of

STS

Fig.

of

AVD

with

RVI

-LRB-

a

-RRB-

Osmolality

was

increased

from

to

mosmol/kg-H

at

time

zero

-LRB-

arrow

-RRB-

with

μM

STS

applied

-LRB-

at

arrowheads

-RRB-

either

h

before

-LRB-

-RRB-

h

before

-LRB-

-RRB-

at

the

same

time

as

-LRB-

STS-HYPER

-RRB-

or

h

after

-LRB-

-RRB-

the

start

of

hypertonic

stress

-LRB-

b

-RRB-

Total

AVD

computed

from

the

overall

volume

changes

shown

in

-LRB-

a

-RRB-

minus

the

initial

amount

of

passive

cell

shrinkage

in

the

first

min

of

hypertonic

stress

-LRB-

i.e.

volume

at

min

minus

volume

at

min

-RRB-

STS-ISO

refers

to

the

AVD

observed

under

isotonic

conditions

n

for

each

experimental

series

Next

the

effects

of

STS

on

activation

of

the

HICC

were

tested

This

was

done

by

increasing

osmolality

at

various

times

relative

to

STS

application

As

shown

in

Fig.

-LRB-

STS-HYPER

-RRB-

STS

had

no

significant

short-term

effect

on

activation

of

the

HICC

when

given

together

with

hypertonic

stress

i.e.

the

peak

activation

of

HICC

current

was

virtually

unaffected

for

min

by

STS

applied

simultaneously

with

hypertonic

stress

This

finding

fits

with

the

observation

that

HICC

activation

appears

to

occur

on

a

considerably

faster

time

scale

than

the

initiation

of

AVD

does

In

this

respect

it

is

also

of

note

that

even

with

STS

applied

h

ahead

of

hypertonicity

-LRB-

-RRB-

the

HICC

was

still

activated

to

a

level

approximately

half

of

the

control

value

i.e.

to

pA/pF

With

STS

given

h

before

hypertonic

stress

-LRB-

-RRB-

HICC

currents

were

almost

completely

inhibited

-LRB-

pA/pF

n

-RRB-

Fig.

of

STS

on

HICC

activation

-LRB-

a

-RRB-

HICC

currents

in

the

absence

-LRB-

Control

-RRB-

or

presence

of

STS

which

was

applied

simultaneously

with

hypertonic

stress

-LRB-

STS-HYPER

-RRB-

h

ahead

of

hypertonic

stress

-LRB-

-RRB-

or

h

ahead

of

hypertonic

stress

-LRB-

-RRB-

Representative

traces

of

to

experiments

-LRB-

b

-RRB-

Typical

I/V-relationships

under

these

conditions

-LRB-

c

-RRB-

Summary

of

HICC

current

densities

obtained

under

the

four

different

conditions

shown

in

-LRB-

a

-RRB-

Whenever

studied

in

a

quantitative

way

HICCs

have

been

proven

to

be

the

main

mechanism

of

the

RVI

of

a

given

system

-LSB-

-RSB-

This

also

holds

for

HeLa

cells

in

which

the

relative

contribution

of

HICC

activity

and

H

antiport

to

RVI

is

approximately

-LRB-

with

no

detectable

contribution

of

K

Cl

symport

at

all

-RRB-

-LSB-

-RSB-

This

raises

the

question

of

whether

the

apparent

interference

of

hypertonic

stress

with

AVD

may

occur

via

activation

of

the

HICC

In

the

experiments

shown

in

Fig.

osmolality

was

increased

from

to

mosmol/kg-H

at

time

zero

-LRB-

arrow

-RRB-

Following

min

of

hypertonic

stress

μM

flufenamic

acid

-LRB-

FFA

an

established

blocker

of

the

HICC

in

HeLa

cells

-LSB-

-RSB-

-RRB-

STS

-LRB-

μM

-RRB-

or

FFA

plus

STS

were

then

applied

-LRB-

arrowhead

-RRB-

As

expected

FFA

significantly

blunted

the

progress

of

RVI

presumably

by

inhibition

of

the

HICC

The

volume

change

from

to

min

-LRB-

volume

at

min

minus

volume

at

min

-RRB-

was

in

the

presence

of

FFA

in

the

control

experiments

on

the

other

hand

the

min

volume

change

due

to

RVI

equalled

-LRB-

n

P

Fig.

Control

-RRB-

Of

note

STS

led

to

a

pronounced

decrease

in

cell

volume

of

-LRB-

Fig.

STS

-RRB-

and

the

effects

of

FFA

plus

STS

were

additive

resulting

in

a

decrease

in

cell

volume

of

-LRB-

P

Fig.

STS+FFA

-RRB-

Experiments

with

mM

borate

-LRB-

-RRB-

as

an

alternative

blocker

of

the

HICC

-LSB-

-RSB-

yielded

virtually

identical

results

With

the

min

volume

change

equalled

-LRB-

Fig.

-RRB-

and

again

the

effects

of

HICC

blockage

and

STS

-LRB-

volume

change

of

P

-RRB-

were

additive

-LRB-

Fig.

STS

-RRB-

Fig.

of

HICC

channel

blockers

and

STS

on

RVI

and

AVD

-LRB-

a

-RRB-

Osmolality

was

increased

from

to

mosmol/kg-H

at

time

zero

-LRB-

arrow

-RRB-

for

all

groups

μM

flufenamic

acid

-LRB-

FFA

-RRB-

STS

or

FFA

plus

STS

were

applied

at

t

min

of

hypertonic

stress

-LRB-

arrowhead

-RRB-

as

indicated

-LRB-

b

-RRB-

Summary

of

the

volume

changes

from

to

min

-LRB-

volume

at

min

minus

volume

at

min

-RRB-

observed

in

the

experiments

shown

in

-LRB-

a

-RRB-

and

two

additional

experiments

in

which

mM

borate

-LRB-

-RRB-

was

used

as

a

blocker

of

HICC

channels

alone

or

in

combination

with

STS

n

for

each

experimental

condition

Significantly

different

from

Control

with

P

significantly

different

from

STS

with

P

and

P

respectively

It

may

be

argued

that

due

to

the

limited

specificity

of

FFA

and

the

effects

of

both

compounds

described

above

may

be

due

to

an

inhibition

of

volume-sensitive

outwardly

rectifying

-LRB-

VSOR

-RRB-

Cl

channels

-LRB-

see

-LSB-

-RSB-

for

review

-RRB-

rather

than

blockage

of

the

HICC

In

an

additional

series

of

measurements

however

it

was

found

that

even

with

application

of

μM

DCPIB

-LRB-

-LRB-

2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl

-RRB-

oxybutyric

acid

-RRB-

an

effective

blocker

of

the

VSOR

Cl

channel

-LSB-

-RSB-

an

RVI

value

of

-LRB-

when

added

at

t

min

n

-RRB-

was

obtained

this

is

virtually

identical

to

the

value

of

observed

under

control

conditions

-LRB-

see

above

-RRB-

Furthermore

μM

DCPIB

inhibited

VSOR

currents

in

HeLa

cells

by

-LRB-

n

-RRB-

whereas

it

did

not

exhibit

an

effect

on

HICC

currents

at

all

-LRB-

n

Wehner

and

Numata

unpublished

results

-RRB-

These

data

strongly

imply

that

even

in

early-stage

apoptosis

HICC

activation

is

an

effective

mechanism

of

RVI

in

HeLa

cells

Furthermore

they

provide

additional

strong

evidence

for

the

functional

interplay

of

AVD

and

RVI

In

this

regard

the

apparent

lack

of

RVI

in

HeLa

cells

undergoing

AVD

-LRB-

Fig.

-RRB-

could

be

accounted

for

as

follows

In

early-stage

apoptosis

-LRB-

say

within

h

after

STS

application

-RRB-

the

AVD

process

surpasses

the

RVI

process

which

involves

HICC

activity

whereas

in

the

later

stage

of

apoptosis

the

RVI

process

is

suppressed

because

of

inhibition

of

the

HICC

by

STS

Finally

we

characterized

the

interplay

between

HICC

activation

and

STS-induced

cell

death

and

apoptosis

As

determined

by

an

MTT

assay

-LRB-

Fig.

-RRB-

a

activity

assay

-LRB-

Fig.

-RRB-

and

an

apoptosis

assay

measuring

nucleosome-bound

DNA

-LRB-

Fig.

-RRB-

increasing

osmolality

from

mosmol/kg-H

to

and

mosmol/kg-H

led

to

significant

and

parallel

reductions

of

both

cell

death

and

apoptosis

observed

h

after

STS

application

the

maximal

effect

was

observed

at

mosmol/kg-H

and

above

This

is

a

surprising

finding

because

based

on

what

has

been

reported

so

far

concerning

the

interplay

of

cell

volume

and

apoptosis

one

might

expect

a

synergistic

effect

of

cell

shrinkage

on

the

induction

of

programmed

cell

death

-LSB-

-RSB-

Furthermore

FFA

and

almost

completely

blocked

these

effects

the

fact

providing

strong

further

evidence

that

it

is

the

activation

of

the

HICC

that

is

actually

rescuing

HeLa

cells

from

apoptosis

Fig.

from

STS-induced

apoptosis

by

hypertonic

stimulation

and

inhibition

of

rescue

by

HICC

blockers

-LRB-

a

-RRB-

Cell

death

observed

h

after

STS

treatment

at

various

osmolalities

and

at

mosmol/kg-H

with

μM

FFA

or

mM

as

indicated

-LRB-

n

-RRB-

-LRB-

b

-RRB-

Apoptosis

monitored

as

activity

induced

by

exposure

to

STS

at

various

osmolalities

and

when

FFA

or

was

also

applied

at

mosmol/kg-H

-LRB-

n

-RRB-

-LRB-

c

-RRB-

DNA

fragmentation

analyzed

with

a

nucleosomal

ELISA

assay

after

a

exposure

to

STS

at

various

osmolalities

and

when

FFA

or

was

also

applied

at

mosmol/kg-H

-LRB-

n

-RRB-

To

insure

that

the

above

measurements

were

not

corrupted

by

the

necrosis

of

cells

propidium

iodide

staining

was

performed

Propidium

iodide

is

known

to

selectively

bind

to

cell

nuclei

and

because

it

does

not

permeate

intact

cell

membranes

it

provides

a

reliable

measure

for

the

necrosis

and

late

apoptosis

of

cells

Following

a

incubation

the

percentages

of

necrotic

cells

were

-LRB-

for

mosmol/kg-H

-RRB-

-LRB-

for

mosmol/kg-H

STS

-RRB-

-LRB-

for

mosmol/kg-H

STS

-RRB-

-LRB-

for

mosmol/kg-H

STS

-RRB-

and

-LRB-

for

mosmol/kg-H

STS

n

for

each

condition

-RRB-

It

is

concluded

from

these

experiments

that

STS

at

μM

is

an

effective

and

reliable

tool

for

the

induction

of

apoptosis

in

HeLa

cells

as

reported

also

in

an

earlier

study

from

this

laboratory

-LSB-

-RSB-

It

may

be

argued

that

hypertonic

stress

-LRB-

and

HICC

activation

-RRB-

just

delays

rather

than

prevents

STS-induced

cell

death

in

HeLa

cells

As

propidium

iodide

staining

after

h

of

STS

exposure

revealed

however

the

necrosis

or

late

apoptosis

of

cells

was

for

mosmol/kg-H

and

for

mosmol/kg-H

Clearly

even

under

these

harsh

experimental

conditions

-LRB-

i.e.

stimulation

with

STS

for

day

-RRB-

hypertonicity

rescued

HeLa

cells

from

death

to

a

significant

degree

-LRB-

P

n

-RRB-

In

the

last

series

of

measurements

the

timing

of

apoptosis

induction

by

STS

versus

hypertonic

stress

was

analysed

As

depicted

in

Fig.

increasing

osmolality

from

to

mosmol/kg-H

rescued

HeLa

cells

from

apoptosis

induced

by

application

of

STS

if

this

maneuver

was

performed

h

before

-LRB-

-RRB-

h

before

-LRB-

-RRB-

or

at

the

same

time

as

the

application

of

STS

-LRB-

STS-HYPER

-RRB-

Moreover

even

with

STS

given

h

ahead

of

hypertonic

stress

apoptotic

cell

death

was

reduced

to

some

of

the

isotonic

control

value

-LRB-

Fig.

-RRB-

Of

note

the

overall

profiles

of

the

effects

of

the

timing

of

STS

application

versus

hypertonic

stress

on

cell

death

and

apoptosis

as

shown

in

the

figure

are

undistinguishable

from

those

observed

on

AVD

-LRB-

see

Fig.

-RRB-

as

well

as

on

HICC

activation

-LRB-

Fig.

-RRB-

Fig.

of

HICC

activation

with

STS-induced

apoptosis

For

the

control

STS

was

applied

for

h

under

isotonic

conditions

-LRB-

STS-ISO

-RRB-

For

the

other

groups

it

was

applied

h

before

-LRB-

-RRB-

h

before

-LRB-

STS

-RRB-

at

the

same

time

as

-LRB-

STS-HYPER

-RRB-

h

after

-LRB-

-RRB-

or

h

after

-LRB-

-RRB-

osmolality

was

increased

from

to

mosmol/kg-H

Apoptosis

was

monitored

as

a

decrease

of

cell

viability

-LRB-

a

n

to

-RRB-

activity

expressed

as

relative

fluorescence

units

-LRB-

FU

b

n

and

-RRB-

and

DNA

fragmentation

measured

in

an

enzyme-linked

-LRB-

ELISA

-RRB-

assay

as

absorbance

values

-LRB-

OD

optical

density

c

n

-RRB-

It

has

long

been

known

that

AVD

occurs

in

parallel

with

a

loss

of

cellular

K

and

a

gain

of

cellular

-LSB-

-RSB-

To

some

extent

these

effects

might

reflect

reduced

activity

of

the

K

ATPase

-LSB-

-RSB-

Nevertheless

if

the

activation

of

the

HICC

were

triggered

as

a

consequence

of

conductive

K

export

and

AVD

this

would

lead

to

very

similar

results

Also

of

interest

in

this

respect

is

a

recent

study

on

human

leukemia

cells

reporting

two

distinct

phases

during

STS-induced

apoptosis

an

initial

phase

in

which

cellular

K

and

Cl

decreased

in

parallel

followed

by

a

second

phase

in

which

there

was

a

further

decrease

in

cellular

K

and

an

enormous

increase

in

cellular

-LRB-

accompanied

by

a

rise

in

cellular

Cl

-RRB-

-LSB-

-RSB-

Of

note

cells

were

shown

to

express

HICC

currents

-LSB-

-RSB-

Thus

the

ionic

scenario

described

could

also

reflect

the

interplay

of

HICC

activation

and

AVD

as

proposed

in

the

present

study

In

rat

hepatocytes

mosmol/kg-H

was

insufficient

to

induce

apoptosis

but

it

sensitized

cells

to

cell

death

whereas

mosmol/kg-H

functioned

as

an

effective

apoptosis

inducer

per

se

-LSB-

-RSB-

Since

rat

hepatocytes

express

-LRB-

amiloride-sensitive

-RRB-

HICC

currents

-LSB-

-RSB-

it

would

be

interesting

to

examine

the

degree

to

which

blockage

of

the

HICC

might

interfere

with

the

induction

of

cell

death

in

this

system

Conclusions

Taken

together

our

results

indicate

that

hypertonic

stress

caused

a

significant

reduction

in

the

staurosporine

-LRB-

STS

-RRB-

induced

apoptosis

of

HeLa

cells

When

analysing

the

functional

interplay

between

apoptotic

volume

decrease

-LRB-

AVD

-RRB-

and

regulatory

volume

increase

-LRB-

RVI

-RRB-

through

the

use

of

different

activation

protocols

and

effective

blockers

the

activation

of

hypertonicity-induced

cation

channels

-LRB-

HICCs

-RRB-

could

be

identified

as

the

molecular

mechanism

by

which

HeLa

cells

are

actually

rescued

from

STS-induced

apoptosis

in

hypertonic

conditions

The

precise

mechanism

by

which

HICC

activation

inhibits

AVD

and

apoptosis

remains

to

be

elucidated

Nevertheless

the

results

reported

herein

demonstrate

a

novel

role

of

the

HICC

in

the

induction

of

programmed

cell

death

They

are

also

in

perfect

agreement

with

a

recent

study

in

which

inhibition

of

the

HICC

was

found

to

sensitize

HeLa

cells

to

shrinkage-induced

apoptosis

-LSB-

-RSB-

We

have

only

begun

to

understand

the

role

of

the

HICC

in

these

processes

-LSB-

-RSB-

and

the

general

relevance

of

the

channel

in

cell

proliferation

and

apoptosis

remains

a

question

of

considerable

interest

O O O O O O O B B I O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O

hela cells
cell shrinkage
volume regulation
apoptosis


Effect of disconnecting the orbital prefrontal cortex from the nucleus accumbens core on inter-temporal choice behaviour : A quantitative analysis Previous experiments showed that destruction of the orbital prefrontal cortex -LRB- OPFC -RRB- or the nucleus accumbens core -LRB- AcbC -RRB- in rats altered choice between two delayed food reinforcers . Application of a quantitative model of inter-temporal choice suggested that lesions of either structure increased the delay-dependent degradation of reinforcer value -LRB- delay discounting -RRB- ; destruction of the OPFC -LRB- but not the AcbC -RRB- also increased the relative value of the larger reinforcer . This experiment examined the effect of disconnecting the OPFC from the AcbC on inter-temporal choice . Rats received excitotoxin-induced contralateral lesions of the OPFC and AcbC -LRB- disconnection -RRB- , severing of the anterior corpus callosum -LRB- callosotomy -RRB- , a combined lesion -LRB- disconnection + callosotomy -RRB- or sham lesions . They were trained in a discrete-trials progressive delay schedule to press levers A and B for a sucrose solution . Responses on A delivered 50 μl of the solution after a delay dA ; responses on B delivered 100 μl after a delay dB . dB increased across blocks of trials ; dA was manipulated across phases of the experiment . Indifference delay , dB -LRB- 50 -RRB- -LRB- value of dB corresponding to 50 % choice of B -RRB- , was estimated for each rat in each phase , and linear indifference functions -LRB- dB -LRB- 50 -RRB- vs. dA -RRB- were derived . The disconnection + callosotomy group showed a lower intercept of the indifference function -LRB- implying a higher rate of delay discounting -RRB- than the sham-lesioned group ; the disconnection group showed a similar but less robust effect , whereas the callosotomy group did not differ significantly from the sham-lesioned group . The results suggest that OPFC -- AcbC connections are involved in delay discounting of food reinforcers , but provide no evidence for an involvement of OPFC -- AcbC connections in regulating sensitivity to reinforcer size . 1 Introduction It is widely accepted that the capacity of a reinforcer to exert control over operant behaviour is a direct function of its size and an inverse function of the delay that precedes its delivery . These two principles are placed in mutual opposition in inter-temporal choice schedules , in which the subject is required to choose between two reinforcers that differ along both dimensions . For example , the subject may be confronted with a choice between a smaller reinforcer , A , of size qA , delivered after a short delay , dA , and a larger reinforcer , B , of size qB , delivered after a longer delay , dB . Inter-temporal choice schedules have provided valuable insights into the behavioural and neurobiological bases of ` delay discounting ' , the hypothetical process whereby the efficacy or ` value ' of a reinforcer decays as a function of delay . Recent work has implicated the core of the nucleus accumbens -LRB- AcbC -RRB- -LSB- 1,2,6,8 -RSB- and the orbital region of the prefrontal cortex -LRB- OPFC -RRB- -LSB- 23,26,42 -RSB- in inter-temporal choice behaviour . However , the interpretation of results obtained using these schedules is complicated by the fact that magnitude and delay of reinforcement are generally manipulated simultaneously . Therefore it is often unclear whether the effect of an intervention on inter-temporal choice behaviour has been brought about by a change in the rate of delay discounting , by a change in the organism 's sensitivity to relative reinforcer size , or both -LSB- 8,19,31 -RSB- . One approach to overcoming this problem is the application of null equations derived from quantitative models of inter-temporal choice -LSB- 19,29 -- 31 -RSB- . For example , according to one such model -LSB- 19 -RSB- , the overall value of a reinforcer , V , is determined by the multiplicative combination of two hyperbolic expressions that define the effects of delay and magnitude upon reinforcer value : where K is the delay-discounting parameter -LSB- 29 -RSB- and Q is a parameter expressing sensitivity to reinforcer size -LSB- 19 -RSB- . Faced with a choice between two reinforcers , A and B , the organism is presumed to select the reinforcer with the higher value . However , by experimentally manipulating the sizes and delays of the two reinforcers , it is possible to establish a state of ` indifference ' , in which A and B are selected with equal frequency . Indifference between A and B is taken to imply equality of value , i.e. , VA = VB . Using Eq . -LRB- 1 -RRB- to define VA and VB and solving for dB -LRB- 50 -RRB- -LRB- the delay to reinforcer B at the point of indifference -RRB- , yields the following null equation : in which dB -LRB- 50 -RRB- is linearly related to dA . By examining the effect of an intervention on this relation , it is possible to deduce whether the intervention has affected the organism 's rate of delay discounting , its sensitivity to reinforcer size , or both . Since K makes no contribution to the slope of the function , a change in slope implies a change in sensitivity to reinforcer size , whereas a change in intercept in the absence of a concomitant change in slope implies a change in the rate of delay discounting -LSB- 19 -RSB- . Destruction of the OPFC was found to increase the slope of the linear indifference function , indicating that the lesion affected sensitivity to reinforcer size ; the lack of a concomitant increase in the intercept suggested that the rate of delay discounting had also been increased -LSB- 26 -RSB- -LRB- see Section 4 for further explanation -RRB- . In contrast , lesions of AcbC reduced the intercept without significantly altering the slope of the function , implying a selective effect on delay discounting -LSB- 2 -RSB- . While these observations implicate both the OPFC and the AcbC in delay discounting , they leave unanswered the question of whether the two structures play independent roles in delay discounting , or whether they contribute to an integrated mechanism regulating inter-temporal choice behaviour . The present experiment was intended to address this question : the effect of functional disconnection of the OPFC and AcbC on inter-temporal choice behaviour was examined using the linear indifference relation epitomised by Eq . -LRB- 2 -RRB- . The principal anatomical link between the OPFC and the AcbC is an ipsilateral excitatory glutamatergic corticofugal pathway , which is believed to comprise one link in a cortico-striato-thalamo-cortical circuit . Inhibitory γ-aminobutyric acid -LRB- GABA -RRB- ergic efferents from the AcbC project to the internal pallidum and substantia nigra pars reticulata . These structures in turn send inhibitory projections to diencephalic structures , whose excitatory corticopetal projections complete the circuit . In order to effect functional disconnection of the OPFC and AcbC without totally ablating either structure , a ` disconnection lesion ' -LSB- 12,14 -RSB- was employed , which consisted of unilateral excitotoxic destruction of the OPFC in one hemisphere and of the AcbC in the other hemisphere . As recent evidence indicates that transcallosal fibres may make a significant functional contribution to cortico-striatal connections -LSB- 9 -RSB- , the effects of callosotomy alone , and callosotomy combined with the disconnection lesion were also examined . 2 Methods The experiment was carried out in accordance with UK Home Office regulations governing experiments on living animals . 2.1 Subjects Sixty experimentally naive female Wistar rats -LRB- Charles River UK -RRB- approximately 4 months old and weighing 250 -- 300 g at the start of the experiment were used . They were housed individually under a constant cycle of 12 h light and 12 h darkness -LRB- light on 06:00 -- 18:00 h -RRB- , and were maintained at 80 % of their initial free-feeding body weights throughout the experiment by providing a limited amount of standard rodent diet after each experimental session . Tap water was freely available in the home cages . 2.2 Surgery Anaesthesia was induced with halothane -LRB- 4 % in oxygen -RRB- , and the rat positioned in a stereotaxic apparatus -LRB- David Kopf -RRB- , with the upper incisor bar set 3.3 mm below the inter-aural line . Anaesthesia was maintained with 2 % halothane in oxygen during surgery . Holes were drilled in the skull for introduction of a microinjection cannula or leucotome -LRB- see below -RRB- . Disconnection : sixteen rats received unilateral lesions of the OPFC and the contralateral AcbC -LRB- the sides being counterbalanced across rats -RRB- . The following coordinates -LRB- mm , measured from bregma -RRB- were used to locate the OPFC : site -LRB- i -RRB- : AP +3.7 , L ± 1.2 , DV − 4.8 ; site -LRB- ii -RRB- : AP +3.7 , L ± 2.8 , DV − 4.4 . The coordinates for the AcbC were : AP +1.2 , L ± 1.8 , V − 7.1 . Injections were given via a 0.3-mm diameter cannula connected by a polyethylene tube to a 10-μl Hamilton syringe . In each site , 0.5 μl of a 0.1-M solution of quinolinic acid -LRB- 2,3-pyridinedicarboxylic acid -RRB- in phosphate-buffered 0.9 % NaC1 -LRB- pH 7.0 -RRB- was injected at a rate of 0.1 μl per 15 s . The cannula was left in position for 3 min after completion of the injection . Callosotomy : 15 rats underwent a midline leucotomy in order to sever the anterior corpus callosum . The leucotome , similar to that described by Gold et al. -LSB- 17 -RSB- , was constructed from a 1-μl Hamilton microsyringe . A curved wire could be extruded from the tip of the syringe needle ; when fully extended , the wire projected approximately 3 mm in the AP plane -LRB- i.e. at right angles to the needle -RRB- . Two midline cuts were made : -LRB- i -RRB- the tip of the needle was positioned at AP 0.0 , DV − 5.0 , the wire extruded in a rostral direction , and the tip slowly raised to DV − 1.0 ; the wire was then retracted into the needle , and the needle was withdrawn from the brain ; -LRB- ii -RRB- the tip of the needle was repositioned at AP +2.0 , DV − 4.0 and the procedure repeated . Disconnection + callosotomy : 15 rats underwent both the disconnection and callosotomy procedures described above . Sham lesion : fourteen rats underwent the same surgical procedures as the disconnection-lesioned group , except that the vehicle solution alone was injected into the target sites . 2.3 Apparatus The rats were trained in standard operant conditioning chambers -LRB- CeNeS Ltd. , Cambridge , UK -RRB- of internal dimensions 25 cm × 25 cm × 22 cm . One wall of the chamber contained a recess into which a peristaltic pump could deliver a 0.6 M sucrose solution . Two apertures situated 5 cm above and 2.5 cm to either side of the recess , through which motor-operated retractable levers could be inserted into the chamber . The levers could be depressed by a force of approximately 0.2 N . A 2.8-W lamp was mounted 2.5 cm above each lever ; a third lamp was mounted 10 cm above the central recess . Six red light-emitting diodes were mounted in a row , 4 cm apart , 5 cm above the levers . The operant chamber was enclosed in a sound-attenuating chest ; masking noise was generated by a rotary fan . An Acorn microcomputer programmed in Arachnid BASIC -LRB- CeNeS Ltd. , Cambridge , UK -RRB- , located in an adjoining room , controlled the schedules and recorded the behavioural data . 2.4 Behavioural training Two weeks after surgery , the food-deprivation regimen was introduced and the rats were gradually reduced to 80 % of their free-feeding body weights . They were then trained to press two levers -LRB- A and B -RRB- for sucrose reinforcement , and were exposed to a discrete-trials continuous reinforcement schedule in which the two levers were presented in random sequence for three sessions . After this initial training , they underwent daily training sessions under the discrete-trials delayed reinforcement schedule for the remainder of the experiment . Each experimental session consisted of six blocks of six trials , except in phases 4 and 5 when sessions consisted of five blocks . The trials were 90 s in duration , with the exception of phase 5 , in which the duration was increased to 120 s in order to accommodate the long delay to reinforcement -LRB- see below -RRB- . The six blocks were signalled by illumination of the six light-emitting diodes : in block 1 the first -LRB- left-most -RRB- diode was illuminated , in block 2 the first and second diodes were illuminated , and so on . The first two trials of each block were forced-choice trials in which each lever was presented alone in random sequence . The other four trials were free-choice trials in which both levers were presented . The beginning of each trial was signalled by illumination of the central light above the reinforcer recess . After 2.5 s the lever or levers -LRB- depending on the type of trial -RRB- were inserted into the chamber . When a lever-press occurred , the lever -LRB- s -RRB- were withdrawn , the central light was extinguished , and the light located above the lever that had been depressed was illuminated . This light remained illuminated until the delivery of the reinforcer , and was then extinguished . The chamber remained in darkness until the start of the following trial . If no lever-press occurred within 5 s of the lever -LRB- s -RRB- being inserted , the lever -LRB- s -RRB- were retracted and the central light extinguished . -LRB- This seldom happened except during the first few training sessions . -RRB- A response on lever A initiated a fixed delay dA , following which 50 μl of the 0.6 M sucrose solution was delivered . A response on lever B initiated a variable delay dB , after which 100 μl of the same sucrose solution was delivered . The positions of levers A and B -LRB- left vs. right -RRB- were counterbalanced across subjects . The experiment consisted of six phases , in which the value of dA was set at 1 , 2 , 4 , 8 , 12 and 0.5 s , respectively . In each phase , the value of dA was held constant . In each session the value of dB was set equal to dA in the first block of trials . In subsequent blocks dB was increased in increments of 75 % . In phases 4 and 5 , when dA was 8 and 12 s , respectively , computing five increments of 75 % would have generated a value of dB that was longer than the duration of a trial in the sixth block of trials ; therefore the number of blocks was limited to five in these phases . The first phase continued for 100 sessions , phase 2 for 50 sessions , and the remaining phases for 40 sessions . Experimental sessions were carried out 7 days a week , at the same time each day , during the light phase of the daily cycle -LRB- between 08:00 and 14:00 h -RRB- . 2.5 Histology At the end of the behavioural experiment , the rats were deeply anaesthetised with sodium pentobarbitone , and perfused transcardially with 0.9 % sodium chloride , followed by 10 % formol saline . The brains were removed from the skulls and fixed in formol saline for 1 week . Forty micrometer coronal sections were taken through the regions of the OPFC and AcbC -LRB- approximately from AP +5.0 to AP 0.0 -RRB- using a freezing microtome . 2.5.1 Cresyl violet staining The procedure was similar to that described previously -LSB- 26 -RSB- . Alternate sections were mounted on chrome-gelatine-coated slides and air dried , hydrated by successive immersion in 95 , 70 and 50 % ethanol , stained in 0.25 % cresyl violet for 2 min at room temperature , dehydrated by successive immersion in 50 , 70 , 95 , and 100 % ethanol and xylene , and mounted with DPX . 2.5.2 Immunocytochemistry In the other sections neurone-specific nuclear protein -LRB- NeuN -RRB- was labelled as described by Jongen-Relo and Feldon -LSB- 21 -RSB- . Our protocol has been described elsewhere -LSB- 2 -RSB- . Briefly , freshly sliced sections were rinsed in 0.1 M phosphate buffered saline -LRB- PBS -RRB- and placed in 0.5 % H2O2 in PBS for 30 min . After twice rinsing in PBS , they were placed for 1 h in a blocking solution -LRB- 10 % normal horse serum -LSB- Vector Laboratories , Peterborough , UK -RSB- , 1 % bovine serum albumin -LSB- BSA , Sigma -- Aldrich , Gillingham , UK -RSB- and 0.3 % Triton X-100 -LSB- Sigma -- Aldrich -RSB- in PBS -RRB- . They were incubated for 48 h at 4 °C with the primary antibody -LRB- monoclonal mouse anti-NeuN serum -LSB- 1:5000 , Chemicon , Chandlers Ford , UK -RSB- in 1 % normal horse serum , 1 % BSA and 0.3 % Triton X-100 in PBS -RRB- , washed twice in PBS , and incubated for 2 h at room temperature in biotinylated horse antimouse serum -LRB- Vector Laboratories -RRB- -LRB- 1:1000 in 1 % BSA and 0.3 % Triton X-100 in PBS -RRB- . After further rinsing in PBS , they were placed for 2 h in avidin -- biotin -- horseradish peroxidase complex -LRB- 1:200 , ABC-Elite , Vector Laboratories -RRB- in PBS . After two further rinses in PBS , they were placed in a chromagen solution -LRB- 0.05 % diaminobenzidine -LSB- Sigma -- Aldrich -RSB- and 0.01 % H2O2 -LSB- Sigma -- Aldrich -RSB- -RRB- for 5 min . The reaction was observed visually and stopped by rinsing in PBS . The sections were floated on to chrome-gelatine-coated slides and mounted with DPX . An investigator who was blind to the behavioural results performed the microscopic examination . Drawings of the area of the lesions were superimposed on the appropriate coronal sections in the stereotaxic atlas of Paxinos and Watson -LSB- 34 -RSB- . 2.6 Data analysis Data from 5 of the 60 rats were discarded . Histological examination revealed two rats with misplaced excitotoxin-induced lesions ; one rat in the disconnection + callosotomy group was discarded because of a failed callosotomy . Data from two further rats were discarded because they showed persistent exclusive responding on one lever . This left 13 rats in the sham-lesioned group , 14 in the disconnection group , 15 in the callosotomy group and 13 in the disconnection + callosotomy group . 2.6.1 Preference functions and linear indifference functions For each rat , the percentage choice of lever B in the free-choice trials -LRB- % B -RRB- was computed for each block of trials from the pooled data from the last 10 sessions of each phase of the experiment . Plots of % B vs. dB were derived for each rat , and the indifference delay -LRB- dB -LRB- 50 -RRB- : the value of dB corresponding to % B = 50 % -RRB- was estimated by linear interpolation between the two delays which fell on either side of % B = 50 % -LRB- i and j -RRB- using the formula : dB -LRB- 50 -RRB- = dB -LRB- i -RRB- + -LRB- -LSB- dB -LRB- j -RRB- - dB -LRB- i -RRB- -RSB- . -LSB- % Bi − 50 -RSB- / -LSB- % Bi − % Bj -RSB- -RRB- -LSB- 39 -RSB- . Plots of dB -LRB- 50 -RRB- vs. dA were obtained for each rat , and linear functions were fitted by the method of least squares ; goodness of fit was expressed as r2 , the proportion of the data variance accounted for by the fitted function . The slope and intercept of the linear indifference functions were analysed by two-factor ANOVA -LRB- presence/absence of disconnection × presence/absence of callosotomy -RRB- followed by multiple comparisons of the lesioned groups with the sham-lesioned group using Dunnett 's test . Linear indifference functions -LRB- dB -LRB- 50 -RRB- vs. dA -RRB- were also derived for the group mean data . The slopes and elevations of these functions were analysed by one-factor ANOVA -LRB- group -RRB- followed by multiple comparisons of the lesioned groups with the sham-lesioned group using Dunnett 's test , as described by Zar -LSB- 43 -RSB- : the slopes were first analysed , and in the absence of significant between-group variation in slope , a common weighted slope value was adopted in order to make comparisons among the elevations . -LRB- ` Elevation ' refers to the y-axis location of the function taking the range of observed data into account , whereas ` intercept ' refers to the intersection of the function with the y-axis location -LSB- 43 -RSB- . -RRB- 2.6.2 Psychophysical analysis of preference functions Logistic functions were fitted to the group mean % B data and the % B data from each rat in each phase of the experiment : % B = 100 / -LRB- 1 + -LSB- dB/dB -LRB- 50 -RRB- -RSB- ɛ -RRB- . This function defines a descending sigmoid curve which is symmetrical in semi-logarithmic co-ordinates ; dB -LRB- 50 -RRB- and ɛ are parameters , dB -LRB- 50 -RRB- being the point of intersection of the logistic curve with the indifference line , and ɛ being the slope of the function . These parameters were used to derive the limen -LRB- -LSB- dB -LRB- 25 -RRB- − dB -LRB- 75 -RRB- -RSB- / 2 , where dB -LRB- 25 -RRB- and dB -LRB- 75 -RRB- are the estimated values of dB corresponding to % B = 25 and % B = 75 , respectively -RRB- , and the index of relative precision , the Weber fraction , was defined as limen/dB -LRB- 50 -RRB- . The Weber fraction was subjected to repeated-measures ANOVA -LRB- phase -RRB- ; as no significant effect of phase was revealed , the Weber fractions were averaged across phases and subjected to two-factor ANOVA -LRB- presence/absence of disconnection × presence/absence of callosotomy -RRB- , as described above . 3 Results 3.1 Behavioural data 3.1.1 Preference functions and linear indifference functions Preference functions -LRB- % B vs. dB -RRB- derived for the four groups in all six phases of the experiment are shown in Fig. 1 -LRB- left-hand graphs -RRB- . In all four groups , preference for lever B declined as a function of the delay to reinforcer B -LRB- dB -RRB- . The horizontal lines in the graphs show the indifference level -LRB- i.e. , % B = 50 -RRB- ; the value of dB at which the preference function crossed this level -LRB- i.e. dB -LRB- 50 -RRB- -RRB- increased as a function of increasing values of dA , reflecting a progressive rightward displacement of the curve . Fig. 2 shows indifference functions -LRB- dB -LRB- 50 -RRB- vs. dA -RRB- for the group mean data . In each group , the linear function accounted for more than 96 % of the variance of the group mean data -LRB- r2 > 0.96 -RRB- . Comparisons were made between the function derived for each of the lesioned groups and the function derived for the sham-lesioned group -LSB- 43 -RSB- . An initial test on the homogeneity of the slopes indicated that there was no significant effect of group upon the slope -LSB- F -LRB- 3,20 -RRB- = 1.8 , P > 0.05 -RSB- , and the common -LRB- weighted -RRB- slope -LRB- 3.16 -RRB- was therefore adopted in statistical comparisons of the elevations of the functions . There was a significant effect of group on the elevation -LSB- F -LRB- 3,23 -RRB- = 24.4 , P < 0.01 -RSB- . Multiple comparisons -LRB- Dunnett 's test -RRB- showed that the elevations of the functions derived for the disconnection and disconnection + callosotomy groups differed significantly from that of the sham-lesioned group -LSB- t -LRB- 9 -RRB- = 4.0 , P < 0.05 , and t -LRB- 9 -RRB- = 5.2 , P < 0.01 , respectively -RSB- , whereas there was no significant difference between the elevations of the callosotomy and sham-lesioned groups -LSB- t -LRB- 9 -RRB- = 0.2 , P > 0.3 -RSB- . Linear indifference functions were also fitted to the data from the individual rats . The group mean values -LRB- + S.E.M. -RRB- of the slope and intercept of the function are shown in Fig. 3 . A two-factor analysis of variance -LRB- callosotomy × disconnection -RRB- showed no significant effect of either factor nor any significant interaction on the slope -LSB- all Fs < 1 -RSB- . There was a significant effect of the disconnection lesion on the intercept -LSB- F -LRB- 1,52 -RRB- = 8.7 , P < 0.005 -RSB- , but no significant effect of the callosotomy -LSB- F < 1 -RSB- and no significant interaction -LSB- F < 1 -RSB- . Multiple comparisons with the sham-lesioned group -LRB- Dunnett 's test -RRB- showed that only the disconnection + callosotomy group differed significantly from the sham-lesioned group . The goodness of fit of the linear function did not vary significantly among the groups -LSB- F < 1 -RSB- ; the function accounted for > 86 % of the data variance for individual rats in all groups -LSB- r2 = 0.865 ± 0.022 -RSB- . 3.1.2 Psychophysical analysis of preference functions The logistic psychometric functions derived for the group mean data -LRB- Fig. 1 , right-hand panels -RRB- accounted for 95 % of the data variance -LRB- r2 > 0.95 -RRB- in all cases . The logistic function could be fitted to 311 of the 330 preference functions obtained for the individual rats in the six phases of the experiment -LRB- 94.2 % -RRB- ; functions could be fitted to the data from all six phases in 48 of the 55 rats . Analysis of variance -LRB- disconnection × callosotomy -RRB- indicated that the goodness of fit was not significantly affected by either the callosotomy -LSB- F < 1 -RSB- or the disconnection lesion -LSB- F -LRB- 1,51 -RRB- = 1.2 , P > 0.2 -RSB- , and there was no significant interaction -LSB- F < 1 -RSB- . The overall mean -LRB- ± S.E.M. -RRB- value of r2 derived from all rats in all phases of the experiment was 0.966 ± 0.005 . There was good agreement between the values of dB -LRB- 50 -RRB- derived from the logistic functions -LRB- dB -LRB- 50 -RRB- logist . -RRB- and those derived by linear interpolation -LRB- dB -LRB- 50 -RRB- interp . -RRB- . The slope of the regression of dB -LRB- 50 -RRB- logist . vs. dB -LRB- 50 -RRB- interp . -LRB- 1.02 ± 0.01 -RRB- did not deviate significantly from unity , and the intercept -LRB- − 0.16 ± 0.31 -RRB- did not deviate significantly from zero ; the correlation -LRB- r -RRB- between the two estimates was 0.971 . The Weber fraction derived from the logistic function was not systematically related to the value of dA . A single-factor analysis of variance with repeated measures -LRB- incorporating data from the 48 rats that generated Weber fractions from all 6 phases -RRB- showed no significant effect of phase -LSB- F -LRB- 5,235 -RRB- = 1.9 , P > 0.05 -RSB- . The Weber fraction was therefore averaged across phases for each rat ; the group mean values -LRB- + S.E.M. -RRB- are shown in Fig. 4 -LRB- left-hand histogram -RRB- . Analysis of variance -LRB- callosotomy × disconnection -RRB- revealed a significant main effect of the disconnection lesion -LSB- F -LRB- 1,51 -RRB- = 6.6 , P < 0.05 -RSB- , but no significant effect of the callosotomy -LSB- F -LRB- 1,51 -RRB- = 2.7 , P > 0.1 -RSB- and no significant interaction -LSB- F < 1 -RSB- . Multiple comparisons -LRB- Dunnett 's test -RRB- did not reveal any significant differences between the lesioned groups and the sham-lesioned group . The group mean values of the slope of the logistic functions -LRB- ɛ -RRB- -LRB- + S.E.M. -RRB- are shown in Fig. 4 -LRB- right-hand histogram -RRB- . Analysis of variance -LRB- callosotomy × disconnection -RRB- showed a significant main effect of the disconnection lesion -LSB- F -LRB- 1,51 -RRB- = 7.2 , P < 0.05 -RSB- , but no significant main effect of the callosotomy -LSB- F < 1 -RSB- and no significant interaction -LSB- F < 1 -RSB- . None of the lesioned groups differed significantly from the sham-lesioned group -LRB- Dunnett 's test -RRB- . 3.2 Histology Examples of the lesions are shown in Fig. 5 . OPFC : injection of quinolinic acid into the OPFC resulted in gliosis and atrophy of the ventral and lateral orbital regions . There was some damage to the medial prefrontal cortex -LRB- medial orbital , infralimbic and prelimbic cortices -RRB- in some rats . The AP extent of the lesion was from about +3.2 to +4.5 ; in no case did the lesion extend caudally as far as the anterior margin of the nucleus accumbens . AcbC : coronal sections showed ventricular dilatation and atrophy in the ventral striatal area . The NeuN labelled sections showed that there was extensive neuronal loss in the area of the AcbC of all lesioned animals , with some neuronal loss in the ventral and medial portions of the caudate-putamen in some animals ; the shell region of the nucleus accumbens was essentially spared . Callosotomy : the corpus callosum was completely severed between AP +0.5 to +1.6 in most animals . There was some sparing of the corpus callosum posteriorly -LRB- caudal to AP +0.5 -RRB- , possibly reflecting the curvature of the leucotome . Destruction of the callosum was generally accompanied by some ventricular dilatation in the vicinity of the lesion , and the mesial surfaces of the cortex overlying the lesion showed some damage . 4 Discussion Injections of quinolinic acid into the OPFC and AcbC produced lesions of similar extent to those seen in previous experiments in which excitotoxins have been used to lesion these structures -LSB- OPFC : 13 , 23 , 25 , 26 , and 42 ; AcbC : 1 , 2 , 3 , 5 , 6 , and 35 -RSB- . In the case of the OPFC lesion , the main area of damage included the ventral and lateral orbital -LRB- VO and LO -RRB- regions . There was also some intrusion into the medial orbital -LRB- MO -RRB- , infralimbic -LRB- IL -RRB- and prelimbic -LRB- PrL -RRB- regions in some animals . According to the subregional classification recommended by Uylings and van Eden -LSB- 40 -RSB- and Kesner -LSB- 22 -RSB- , the lesion embraced the ventral -LRB- VO -RRB- and lateral OPFC -LRB- LO -RRB- , with some involvement of the medial PFC -LRB- MO , IL and PrL -RRB- . In the case of the AcbC lesion , the area of destruction was mainly restricted to the target structure . Some additional damage was inflicted to the ventral portion of the caudate-putamen in some rats ; and in some cases NeuN staining revealed a band of neuronal loss in the medial caudate-putamen adjacent to the lateral ventricle . The mesial shell region of the nucleus accumbens was spared . The callosotomy lesion was generally successful in severing the corpus callosum anterior to the caudal margin of the AcbC . The discrete-trials schedule used in this study was an adaptation of the progressive delay schedule developed by Evenden and Ryan -LSB- 10 -RSB- . The rats in all four groups showed a progressive shift in preference from the larger to the smaller reinforcer as the delay to the larger of the two reinforcers -LRB- dB -RRB- was progressively increased across successive blocks of trials . This is consistent with previous studies that have used this schedule -LSB- 2,6 -- 8,10,11,23,24,26,33,42 -RSB- . As in previous experiments -LSB- 2,23,24,26 -RSB- , we used a geometric progression to determine the values of dB in successive blocks of trials , thereby allowing the range of values of dB to be adapted to the value of dA , which was systematically manipulated across the six phases of the experiment . The resulting preference functions -LRB- relation between % B and dB ; see Fig. 1 -RRB- were used to compute the indifference delays to the larger reinforcer -LRB- dB -LRB- 50 -RRB- -RRB- in each phase . This measure formed the basis of the linear indifference functions -LRB- see below -RRB- . As in previous studies employing Evenden and Ryan 's -LSB- 11 -RSB- protocol -LSB- 2,6 -- 8,10,11,23,24,26,33,42 -RSB- , the preference functions seen in this experiment were characterized by a gradual reduction of % B as a function of dB . As noted by Bezzina et al. -LSB- 2 -RSB- , this is apparently inconsistent with models of inter-temporal choice that are based on the computation of hypothetical ` values ' of reinforcers . Such models generally assume that organisms should invariably select the more highly valued of two mutually exclusive reinforcers -LSB- 18,19 -RSB- , leading to the prediction that the ideal preference function should be a step function , % B falling precipitously from near 100 % to near 0 % around the point at which VA = VB . Bezzina et al. -LSB- 2 -RSB- proposed that the gradual decline in preference generated by progressive delay schedules may represent a discrimination gradient for reinforcer value , and adopted a standard psychophysical approach to analyse the preference functions , an analytical approach that was also used in the present study . As in Bezzina et al. 's -LSB- 2 -RSB- experiment , a two-parameter logistic function adequately described the group mean preference functions -LRB- Fig. 1 , right-hand panels -RRB- and more than 90 % of the preference functions obtained from individual rats . The two parameters of this function define its slope -LRB- ɛ -RRB- and its locus on the abscissa -LRB- dB -LRB- 50 -RRB- -RRB- . Combination of ɛ and dB -LRB- 50 -RRB- allows computation of the Weber fraction , the traditional measure of the relative precision of discrimination -LSB- 15,16,20,28 -RSB- . The data shown in Fig. 4 indicate that ɛ tended to be lower and the Weber fraction higher in the disconnection-lesioned and disconnection + callosotomy groups than in the sham-lesioned group . This resembles Bezzina et al. 's -LSB- 2 -RSB- finding with bilateral AcbC lesions , which induced a robust increase in the Weber fraction , consistent with an impairment of discriminative precision . It appears that functional disconnection of the OPFC from the AcbC had a similar effect on value discrimination as destruction of the AcbC . The locus of the preference function is defined by dB -LRB- 50 -RRB- , the delay to reinforcer B corresponding to indifference between the two reinforcers . According to hyperbolic models of inter-temporal choice -LSB- 19,29 -RSB- , indifference implies equality of the values of the two reinforcers , which provides the basis for deriving the linear function expressed by Eq . -LRB- 2 -RRB- . This equation offers a means of distinguishing between changes in inter-temporal choice behaviour brought about by effects on the hypothetical processes of delay discounting -LRB- K -RRB- and magnitude discounting -LRB- Q -RRB- . The slope of the linear function reflects the physical magnitudes of the two reinforcers -LRB- qA and qB -RRB- and Q ; a change in slope therefore implies a change in Q . The intercept of the function is influenced jointly by Q and K. Therefore , while an increase in Q causes an increase in both the slope and the intercept , an increase in K simply diminishes the intercept . If both parameters are increased , Q 's effect on the intercept is countered by the change in K , whereas its impact on the slope remains unaltered -LSB- 19 -RSB- . In the present experiment , the indifference functions of the four groups did not differ significantly in terms of slope ; however , there were significant between-group differences in the intercept . The disconnection-lesioned group showed a somewhat lower intercept than the sham-lesioned group . This effect was statistically significant when the functions fitted to the group mean data were compared ; however it failed to reach significance when the functions fitted to the data from individual rats in the two groups were compared . A more robust effect on the intercept was seen in the case of the disconnection + callosotomy group , whose intercept was significantly lower than that of the sham-lesioned group , in the case of the functions fitted to the group mean data and also the functions fitted to the individual-subject data . The callosotomy had no significant effect on the intercept . These results indicate that functional disconnection of the AcbC from the OPFC selectively altered the delay discounting parameter , K . The lower intercept seen in the disconnection + callosotomy group implies a higher value of K , in other words , a higher rate of delay discounting , than that of the sham-lesioned group . The fact that callosotomy alone had no discernable effect on performance suggests that ipsilateral cortifugal fibres may be mainly responsible for the functional connection between the OPFC and AcbC ; however , the fact that the combined disconnection + callosotomy lesion had a more robust effect on inter-temporal choice than the conventional lesion suggests that inter-hemispheric connections may also make a subsidiary contribution . The existence of inter-hemispheric cortico-striatal connections has been known for some time -LSB- 32 -RSB- , although their functional importance has only recently been established . Dunnett and his colleagues -LSB- 9,41 -RSB- recently found that a combined disconnection + callosotomy lesion was needed to effect complete functional disconnection of the prefrontal cortex and dorsal striatum in the case of delayed alternation performance . Previous experiments have examined the effects of selective lesions of the OPFC and AcbC on inter-temporal choice behaviour . Kheramin et al. -LSB- 23,26 -RSB- found that bilateral destruction of the OPFC increased the slope of the linear indifference function without altering the intercept , an effect that was attributed to an increase in the rate of delay discounting combined with increased sensitivity to relative reinforcer size -LRB- see also -LSB- 25 -RSB- -RRB- . Cardinal et al. -LSB- 6,8 -RSB- first reported that bilateral lesions of the AcbC rendered rats more ` intolerant ' to delay of reinforcement . This finding was recently confirmed and extended by Bezzina et al. -LSB- 2 -RSB- , using the indifference-equation approach ; these authors reported that destruction of the AcbC lowered the intercept of the linear indifference function without significantly altering the slope , implying an increase in the rate of delay discounting . These findings suggest that both the OPFC and AcbC may contribute to delay discounting . The present results further suggest that delay discounting may be regulated by an integrated mechanism that involves both these structures , and that the integrity of both structures and their connecting fibres may be important for the effective control of behaviour by delayed reinforcers . Interestingly , the present results provide no evidence for an involvement of OPFC -- AcbC connections in regulating sensitivity to reinforcer size . This suggests that while the OPFC may integrate information on multiple features of reinforcers , including both size and delay -LSB- 37,38 -RSB- , its role in delay discounting may be more specifically related to its connections with the AcbC . Data consistent with this notion have recently been obtained using the progressive ratio schedule . Mathematical analysis of performance on this schedule -LSB- 27 -RSB- yields a quantitative index of instantaneous reinforcer value that is sensitive to variations of reinforcer size -LSB- 4,36 -RSB- . This index has been found to be reduced by lesions of the OPFC -LSB- 25 -RSB- but not by lesions of the AcbC -LSB- 3 -RSB- , suggesting that the OPFC , but not the AcbC , may be involved in determining sensitivity to reinforcer size .

O O O O B I I O O B I I O O O O O O O O O O O O O O O B I I O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

orbital prefrontal cortex
nucleus accumbens core
disconnection lesion
delay discounting
rat