Effects of cyclic nucleotides on behaviour, electrocorticogram and cortical acetylcholine release

Pharmacological

Research

Communications,

347

Vol. 7, No. 4, 1975

EFFECTS OF CYCLIC NUCLEOTIDES ON BEHAVIOUR,

ELECTROCORTICOGRAM

AND CORTICAL ACETYLCHOLINE C. Bianchi

L. Beani, Department Department Received

28 May

RELEASE

and A. Bertelli

of Pharmacology, University of Chieti and University of Pisa (Italy) of Pharmacology,

1974

SUMMARY Cyclic

AMP and

lateral

ventricle

whilst

cyclic

suggest

pathways

in

that

GMP reproduces with

in

the

this

derivative,

guinea-pig,

GMP determined

results

associated

the

and reduction

cyclic

These

dibutyryl

of

synchronization whereas

its

caused cortical

cyclic of

into

sedation,

the

E,Co,G,

Acetylcholine

opposite

release,

effects. AMP mimics

Dopamine

stimulation

injected

central

the

Noradrenaline, actions,

corticipetal

which

are

cholinergic

species.

INTRODUCTION Up to (cGMP)

now a clear

in

population

the

C.N.S.

in

homogeneous. (Kakiuchi

Rall,

1970;

et al.,

structure,

Thus,

tissue

by injecting

et al., 1973;

of

3*5*AMP

difficult

1969;

Von Voigtlander

nucleotides

contradictory directly

because

and function of

and chemical Krishna

(CAMP) and 3t59GMP

to assess

concentrations

Moreover, the

role

biochemistry

by physical

Daly,

1973).

has been

its

can be influenced kinds

functional

et al.,

of

1970;

et al., results into

is

one or both stimuli

the

1973; have brain:

cell not

compounds different

Sattin

and

Goldberg been for

reached instance,

Pharmacological

348

the

dibutyryl

derivative

rats,

chickens

1970;

Marley

of

and fowls

(McKean 1972;

ineffective,

On the

neurodepression

and sleep-like

fowls,

the

mimicing in

this

The similarity strengthened of

the

by the

nucleotide

et al,,

1973)

reproduces nergic delli

to brain

cells

from

inhibits the

1974),

1970;

the

we extended

of ascertaining

whether

Meeting Rome,

of

the

following

the

French

to

of catecholamine: of CA is application

al.,

1971;

Anderson

et al.,

1973)

(DA) hyperpolarization, are

Greengard

et

with

choli=

antagonistic al.,

to those

1973;

Ferren=

19721, that

DA stimulates

and telencephalic

experiments

in

this

results and

Italian

carried

out

1973;

Beani

species

with

to mimic

cGMP was really were

and NA

cholinergic

1970,

cAMP was able

of NA or DA and whether Some of

et

which

and Bianchi,

the

rise

CAMP and those

(Greengard

evidence

(Beani

CAMP

1972).

(Siggins

1973;

behaviour

guinea-pig

CAMP gives

cGMP seems to be associated

indirect

gross

et al,,

microionophoretic

events

devoid

the

structures et al,, the

central

aim actions

of any activity.

communicated

at

the

Joint

Societies

of Pharmacology

in

on guinea-pigs

of both

1973.

MATERIALS AND METHODS Experiments weight some of

about the

were 500 g. animals

guide-cannula of

steel

screws

in

the in

Two or three were

submitted

left

ventricle

the

7975

cats,

whereas

actions

(NA) and dopamine

McAfee,

the

the

4,

theophylline-pretreated

of

neurones

by CAMP (Goldberg,

Moving of

that

and cellular

et al.,

effects

finding

In contrast,

promoted

hand, in

7, No.

in

Gessa 19731,

and E.Co.G,

the

noradrenaline

activity

other

Vol.

excitation

1969;

and Nistico,

and ganglionic

respectively.

et al,,

signs

(Marley between

causes

Cohn et al.,

behavioural

species

Communications,

CAMP (DB)

and Nisticb,

and cGMP are

(CA)

Research

frontal

days to

before

the

the

implantation

(Hayden

et al.,

and parietal

bones.

sexes,

experiment of a 1966)

and

The screws

Pharmacological

were

Research

connected

acting

parietal

cannula

in

the

filling

the

cortex

ACh content Finally,

a third cannulas,

temperature

and,

under

urethane

ethyl

On the kept

were

trained

let

in

volumes three

Guanosine

at

E.Co.G.,

followed pl

for

cages

provided

of

the

with

E.C.G.,

rectal

(carotid the

artery)

animals,

and had free

cortical at

least

physiological into 0.07,

the 0.2,

which

access

to food,

experimental

room and to

ACh outflow,

body

temperature

the

nucleotides,

injected

in

drugs

3'5?GMP, by Boehringer

solution,

lateral 0.5

one hour. ventricle.

were 5*AMP,

also used:

Then were

DB was assayed

at

)IM; CAMP and 5*AMP at 0.5 FM;

In a few experiments,

1 and 2 FM were

The following supplied

surgery

1958).

be handled.

and 1.5 PM.

derivative,

day after

and

(Murnaghan,

pressure

anaesthesia.

environment

levels:

cGMP at 0.5

blood

minutes

standard

i.p.>

to the

5-25

dose

only

to record

solution

fifteen

of guinea-pigs,

plastic

in buffered of

every

in

a guide-

The eserinized muscle

open-top

were

dissolved

19701,

dorsal

(1 g/Kg

behaviour,

and E.C.G.

on leech

and second

themselves Normal

ventricle.

was used

1968)

cup was implanted

and Bianchi,

in a few cases,

first

were

(Beani

group

et al.,

electroencephalographic

cup was renewed

assayed

ventricular

(Beani

an epidural

contralateral

epidural

socket

monopolar

animals

349

Vol. 7, No. 4, 1975

valve

for

In other

right

its

to a small

as a terminal

recordings. the

Communications,

Adenosine

and

tested, 3954AMP,

Guanosine

3*5'AMP

and Adenosine,

dibutyryl kindly

Co.

RESULTS Behaviour. A slight increase was sometimes --------and exploratory activity, immediately after in

5-25

minutes

pl. after

However, which

the excitatory all the animals

observed

in

locomotor

DB or CAMP injection

state only lasted showed drowsiness,

for l-2 ataxia,

Pharmacological

350

swaying on their

the

head,

sides.

This

barely

manifest

DB 0.5

PM; CAMP 0.5

to

those

contrary,

Fig.

of

induced

after

Communications,

reduced

muscle

tone

picture

lasted

for

DB 0.07 pM caused

by the

cGMP induced

Research

highest

pM, but symptoms

and tendency about which

7, No.

to

one hour;

particularly

and lowest

a dose-dependent,

Vol.

were doses

prolonged

4,

lay it

evident

1.975

down was after

intermediate of DB, On the increase

in

1. Unipolar E.Co,G. records from the right (r) and left (11, frontal (f) and parietal (p) cortical areas of a guinea-pig: top, control records; middle, 20 min after i.c.v. injection of DB-3?5*AMP 0.2 PM; bottom, 20 min after i.c.v. injection of DB-3*5*AMP 0.5 ).I&! Note the slow-wave synchronized pattern caused by DB at both dose levels.

Pharmacological

Research

locomotor

Communications,

and exploratory

was so strong and showed detected

that

the

stereotyped, in

or Guanosine

gross l-2

Vol.

activity; animals

tried

after

351

1975

1,5

uM the

escape

from

movements.

excitation their

No change

5?AMP 0,5

uM and

(E,Co.G,)

picture

consisted

in

after

cages was Adenosine

uMO

pl)

synchronized

to

2,

to

masticatory

behaviour

4,

after

E,Co.G, The electrocorticographic -----after DB O-07-0.5 FM (in 5-25

Fig.

7, No.

pattern,

similar

that

seen

5-10

a slow

during

minutes

wave,

barbiturate

E.Co.G. records as in Fig. 1. tracing; middle, Top, control synchronization caused by pentobarbital 20 mg/Kg, i.p,; bottom, the reversal of sleep-like E.Co.G. pattern following injection of 3'5'GMP 0.5 JIM. i.c.v.

(3726)

uM

uM

cGMP 0.5

cGMP 1.5

(3622)

(4423)

(6227)

(39+3)

(4829)

(3424)

7

5

(40+3)

7

1st

98+3

10525

loo+1

98+2

100+4

10025

98+1

97+4

2nd

104~4

104~4

lOl+l

10225

10425

103~4

10725

9624

3rd

Pre-treatment collection periods

5'AMP 0.5uM

CAMP 0.5

uM

uM

DB 0.2 in 5 ul

uM

uM

DB 0.2

DB 0.5

uM

DB 0.07

No. of expts

201+12**201+16**

13729"

133+13*

192+11**

142+8**

177+14**

137+13*

119+5

11927 102~8

109+9

11024

9824

78+3*

69+5*

190+16**

14129**

69+5**

58+8**

51+4**

10028

11228

53+6-k*

8828

9323

12228

8th(75)

6928**

66+5*"

11426

7th(60)

after

63~6**

115+5

77+4*

8628

7624"

63+6**

110+7

92L13

102+9 82+5*

6th(45)

Post-treatment periods (minutes

5th(30)

collection

4th(15)

Treatment 4

185~17""

143+10*"

117~6

12528"

8029"

11928

11229

11124

9th(90)

injection)

derivative (DB), 5'AMP and cGMP on cortical ACh release in the 1. Effect of CAMP, its dibutyryl The injection (25 ul, if not otherwise stated) was made in the lateral ventricle guinea-pig. each period lasted 15 minutes. In the first column, at the end of the third collection period; the absolute normal amounts of ACh release (ng/cm2/15 min) are given in brackets. All the other values are the means (+S,E.) of the percentages calculated in each experiment with respect to the first collection period. Student t-test for paired data: Significantly different from the first pre-treatment value, ** , P ( 0.01, * 2 P( 0.05;

Experimental conditions

Table

5 .k 2 P P G 2

E h)

Pharmacological

Research

sedation

(Fig.

minutes,

a time

1-2

substantially

modify

deriving

E,Co.G, the

potent

pM were

pattern,

In

reached

corresponding

was less

Guanosine

nized

The effect

l),

to

owing

from order

the to

animals

the

were DB;

to

most

severe

obtained

with

Even

E,Co.G,, the

acoustic

and

at

30-45

behavioural CAMP, which

pM, Adenosine

cGMP 0.5-1.5

characterized

whether

the

synchronization

with

maximum

5?AMP 0.5

ineffective, the

its

and

PM did

not

by a desynchro=

visual

stimulations

environment,

ascertain

counterpart,

than

353

Vol. 7, No. 4, 7975

The same results

depression, however

Communications,

Pentobarbital

IO-20

excitation

by cGMP had an

was induced

by treating

mg/Kg

then,

i.p.:

cGMP

r

nmeanofinhibition afterDB-3'a.AMP .max.inhibition afterD&?XAMP

001

02

05

dose (IogscaleJpM

Fig.,

3,

Relationship between doses of DB-3?5*AMP, injected i.c.v., and reduction in ACh release, as per cent of the control values; n -m, mean of inhibition during 90 min after treatment (ave= rage of six collection periods, see Table l);o-----• ~ maximum of inhibition, 30 min after treatment,

354

0.5

pM (5 or 25 pl)

As shown changed

in Fig. the

tracing.

Pharmacological

Hesearch

was inJected

into

2, within

slow

reacted

even

to

the

light

the

into

tactile

the

30-45

minutes

them after clearly

(Table

dose-dependent,

The drug, exactly

injected in

arose,

tried

CAMP 0.5YM either slightly

1).

was

two

the

in

of

times

lowest

values

or rose caused

affected

in

above

by DB was

less

(Fig,S).

the

release

25 ~1. effective,

outflow

whilst

unchanged

5*AMP

or else

it. cGMP 0.5-1.5

in ACh release,

its

and intensity

5 pl, did

neurotransmitter

In contrast,

and

was significantly

levels

duration

as it

or three

increased

nucleotide

to walk

The reduction

in a volume

same manner

left

to normal

both

the

7975

stimulations.

and returned

one hour

4,

desynchronized

Acetylcholine release. Cortical ACh release -----I--------------reduced 15 minutes after DB or CAMP, reached at

7, No.

ventricle.

cyclic

a highly

animal

Vol.

lateral

a few minutes

wave E.Co.G.

Concomitantly

Commumcatlons,

,uM produced

as expected

by its

a long-lasting

increase

influence

on behaviour

have

tested.

and

E,Co,G, Neither

Adenosine

nor Guanosine

Body temperature and heart --------__------__-_----------with a time course similar (Table

2).

0.07-0.2 of the

Body

latter

were

animals,

Both

not

as one or three

vanished

30 minutes

tested,

for

after

S'AMP,

and with

body 0,2

temperature

FM) lowered

kept blood

(but

of

not

after

ACh release

the lasted

under

ethyl

constant

at

reduction by DB

pM: higher the

doses

restlessness

CAMP) reduced

but

pressure

hypothermia,

was unaffected

because

minutes

DB O-5 PM, In guinea-pigs at

found

however,

DB and 5tAMP

as early

not

to that

pM caused

uM and by CAMP, 5fAMP and cGMP 0.5

the

after

DB at 0.5

rate,

temperature,

been

heart

injection. for

over

urethane 15-20

rate

The effect one hour anaesthesia

38OC, DB 0.5

by about

of

uM (but

mmHg in

0.5

cGMP

p'M

(5)

a b

;

44

285219

277214

248219

37.3+0.4

m----w

37,420,2

37,5+0.3

--_--200+10*

24624

233+11

-w-e--

37-420-3

36,9&0.3" 1 2 6 + 1 5 +:-'<

._--_--

1 &,+2

1‘&,+6;k" -

37,2,0,2

15

23529

---_--

3

170+6*

230+5

'6)

0.5

PM

37,70.2

S'AMP

fi

240~2

37,6+0,1

250+10

(6)

a b

251~6

37,6+0,2

0.5

FM

(6)

a b

37,6~0,3

PM

pM

(6)

CAMP

0.5

DB

0.2

DB

Controls

a)Temperature OC b)Heart rate/min

Drugs

255+9

37.5+0..3

24026

37,9,0,4

24529

37,4+0,2

138+17"x

36,4+0,3*

270212

28222 1

37,5+0.3

230~6

230~10 37,8+0,2

38.0~0.4

255210

250210 38.0+0,3

37,8+0,1

174+7"" -

36,4~19~

23529

3706~0.3

60

37"7&0,1

1 6 8 -+ 1 4 f
36,3~0,34

225+6*+~

205,5*"

45

treatment

37,2+0,1

after

37,2+0.2

30

Minutes

37.5+0,4 292+19

285~15

235~6

38,1+0,3

245+9

38.0~0.1

198+12*

36.9+0,3*

252212

37.420.2

90

37.8+0,2

226~5

38,120.3

255210

38,120.2

180.t13Jc"

36.5+0.4

256410

37,4-+0.3

75

Effect of CAMP, its dibutyryl derivative (DB), 5PAMP and cGMP, injected in the lateral ventricle, on rectal temperature and heart rate (&S,E.) in the guinea-pig. The injection was performed after two measurements of the basal values (controls), In brackets, the number of experiments. Significantly different from the control values, Student t-test for paired data, *, 6 ( 0,05; **) P( 0.01,

2.

Table

356

Pharmacological

lo-15

minutes.

rate.

After

At

the

DB the

artificial

Research

it

same time,

depth

respiration

Commr/nications,

Vol.

progressively

of anaesthesia was needed

slowed

was so severe

to prevent

7, No.

the

4,

1975

heart

that

animal*s

death,

DISCUSSION The choice

of

the

intraventricular

substances

unable

useful

the

study

of their

as to

their

site

for

problems In our

to cross

experimental

CAMP and cGMP effects

of

the

in

preliminary

trials

injected

in

minutes,

while

within

in

it

15 minutes

that

the

the

5 ~1,

they

always

minutes

after

the

in

their

that

are

easily

spread

the

the

some

and not

over

fourth

blue,

ventricle it

neuronal

on the

hand,

methylene

after

can be inferred

cortical

near

hemispheres:

and ACh release

cortical

30

spaces

structures

an insufficient the

other

subarachnoidal

behaviour

injection,

on the

25 ~1~ Thus,

on deep

and extent

independently

same,

that

to the

in

affected

connected of

the

concerned,

On the

the

claim

other that

depressant protection

compensate reverse

period

surface,

on this

problem As far

a few of

time

when injected

effects

as the

ground

it

enzyme with

more

respect

difficult

would

nucleotides

easily

the

and Nisticb, rate

of action,

adenine

inhibitors

inactivation

diffusion

site

of

is

phosphodiesterase

slower

the

effectiveness

of CAMP (Marley the

of

of DB to diffuse

greater

versus for

its

ability

fowls

action

the

of action.

the

hand, in

with

type

CAMP may explain

the

raises

a 5 ~1 volume. Strictly

is

although

course

(5 or 25 ~1);

reached

spreading

time

was ascertained

acted

fact

the

hardly

system

barrier,

effects,

nearly

used it

nucleotides

administering

of action.

when given

ventricular

for

central

were

volume

for

the blood-brain

conditions

of

injection

route

former to

1972):

compound.

justify

unmask

the

a

in

fact

be expected

of CAMP but

to DB. Therefore,

than

not further

to to

Pharmacological

Research Communications,

arguments that

in

area

are

the

must

be considered.

different not

and

cats

the

(Gessa

et

precursors are

(ii),

the

for

to

is of

1970;

of to

the

well

brain

and

Daly,

guinea-pigs

known

a given

1973;

experiments

nucleotides

their

through

Kodama et

from

can

by the

lead

rats to

the

injection

and

nucleotides

limits the

points of

the

structure,

in of

us from

changes

the

cell

function,

effect

injection

and

on isolated

heart

the

method

of

exerted

intraventricular

the

biological

activities

drawing

definite

conclusions,

emerge

induced

because

in

1972).

inherent

seem to

properties"

chronotropic

applied

al.,

non-cyclic

role

intraventricular

complexity

dissuade

following

et

their

"pharmacological

negative

59AMP directly

(Bertelli

Although

and

physiological

5'AMP after

by CAMP and

most

al.,

may share

instance,

preparations

-

cyclic

unrelated

DB and

by

et

1970)

or derivatives

as shown, (i),

it

results.

Furthermore,

which

all,

357

same neurotransmitter

extension

al,,

of

adenylcyclases

by the

(Krishna

thus,

unexpected

the

stimulated

1973);

First

species

same receptors

al.,

Vol. 7, No. 4, 1975

from

our

the

the

results:

by CAMP depend

corresponding

of

on its

non-cyclic

cyclic compound

is

ineffective; -

in

the

guinea-pig

action:

the

release

decreases.

sensitive body

picture of

is

highly

which

are

signs

reflect

shown

by

barbiturate

The vegetative

temperature,

blood

pressure

and

from

that

Stephenson,

active

in to

an actual increased sedation.

the

those

and

centres higher

far

same kind

synchronized

indeed,

opposite

the

becomes

the

treatment:

not

NA (Marley

cGMP is

in

E.Co.Ci.

to

affect

-

CAMP and DB show

caused

the

appear doses

are

of

sedative

cortical to

be less

required

and heart

ACh

rate,

by intracerebral

to This

injection

1970). guinea-pig caused

enhancement ACh output

and

produces

symptoms

by CAMP. The excitatory of

neuronal

and by

the

activity, awakening

as effect

Pharmacological

358

Thus,

in

the

stimulate Beani acts

guinea-pig,

mainly et al.,

pre-

through

1974).

cGMP appears dopamine It

a cholinergic

remains

by the

The skillful gratefully

Consiglio technical

to mimic

(Anden

work

(Beani

assistance

drugs

and Bedard,

delle of Mr.

7, No.

known

and Bianchi, whether

directly

was supported

Nazionale

Vol.

to be ascertained either

link this

Cotnmunications,

receptors

or post-synaptically,

Acknowledgements: awarded

the

Research

4,

1975

to 1970; cGMP

or indirectly 1971).

by grant Ricerche, A. Giacomelli

No.

720065004,

Rome. is

acknowledged.

REFERENCES Anden N.E. and Bedard P., J. Pharm. Pharmac. 23, 460, 1971. Anderson E.G,, Hans H.L. and Hosli L., Brain Res. 49, 471, 1973. Mechanisms in Beani L. and Bianchi C., In: Drugs and Cholinergic the CNS (Heilbronn E. and Winter A., Eds.) pp.369, Research Institute of National Defence, Stockholm, 1970. l2, 283, 1973. Beani L. and Bianchi C., Neuropharmacology A., Europ. J. Pharmac. 26, 63, Beani L., Bianchi C. and Castellucci 1974 Beani L,, Bianchi C., Santinoceto L. and Marchetti P., Int. J. Neuropharmac. 1, 469, 1968. Bertelli A., Bianchi C. and Beani L., Europ. J. Pharmac. 19, 130, 1972, Cohn M.L., Yamaoka H., Taylor G.H. and Braynack B., Neuropharmacolog> l2, 401, 1973. Daly J.W., Life Sci. l-3, XXIV, 1973, Ferrendelli J.A., Steiner A.L., McDougel D.B. and Kipnis D.M., Biochem. Biophys. Res. Comm., 41, 1061, 1970. Gessa G.L., Krishna G., Forn J., Tagliamonte A. and Brodie B.B., Advances in Biochemical Psychopharmacology 3, 371, 1970. Goldberg N.D., Haddox M.K., Hartle D.K. and Hadden J.W., In: Pharmacology and the future Man. 5th Int. Congress of Proc. Pharmacology, 3, 146, 1973, San Francisco (Karger ed., Basel). GreenPard P., Kebabian J.W. and McAfee D.A., In: Pharmacology and the future Man. Proc. 5th Int, Congress of Pharmacology, 5, 207, 1973, San Francisco (Karger ed., Basel). Hayden J.F., Johnson L.R. and Maickel R.P., Life Sci. 3, 1509, 1966. Kakiuchi S., Rall T.W. and McIlvain M., J. Neurochem. 16, 485, 1969. Kodama T,, Matsukado Y. and Shimira H., Brain Res. 50, 135, 1973, Krishna G., Forn J., Voigt K., Paul M. and Gessa G.L,, Advances in Biochemical Psychopharmacology 3, 155, 1970.

Pharmacological

Research

Communications,

Vol.

7, No.

4,

1975

Marley E. and Stephenson Y,D., In: Catecholamines Muscholl E., Eds.), Springer Verlag, Berlin, Marley E. and Nisticb G., Brit. J. Pharmac. 46, McAfee D.A. and Greengard P., Science 178, 310,

359

(Blaschko H. and 1972, PP. 463. 619, 1972. 1972.

McKean CUM., Peterson N,A. and Raghupathy E., Fed. Proc. 28, 776 abs., 1969. Murnaghan M.F., Nature (London) 182, 317, 1958. Sattin A. and Rall T.W., Molecular Pharmacology 5, 13, 1970. Siggins G-R., Oliver A.P., Hoffer B.J, and Bloom F.E., Science 171, 192, 1971. Von Voigtlander P.F., Boukma S.J. and Johnson G.A., Neuropharmacology l2, 1081, 1973.