Studies on frog brain acetylcholine

Pharmacological Research Communications, VoL 4, No. 2, 1972

STUDIES

ON FROG BRAIN A C E T Y L C H O L I N E

A. G i a m b a l v o , Department School

of

135

G. M a i n a r d i

Pharmacology,

and Department

of

University

a n d G. P e p e u *

University

of

Pharmacology, of Cagliari,

Florence

School

of

Medical Pharmacy,

Italy.

Received 23 June 1972

SUMMARY ranges

ACh l e v e l

between

being

lower

in

spring

the

level

higher

frog

of

of the

to

and early

those

summer t h a n

ACh,

induce

observed

scopolamine

brain

in

frog

(rana

and shows seasonal

and oxotremorine

similar

hand, in the

brain

8 a n d 12 ~ g / g

Pentobarbital ACh l e v e l ,

in the

contrast

variations,

in the changes

i n mammals.

administration with

esculenta)

other

in brain

On t h e

does not the

months.

effect

other

decrease seen

in

vertebrates.

The b r a i n in that includes

the the

of

amphibians

telencephalon

is

telencephalic

The t e l e n c e p h a l i c

cortex

differs

from that

o f mammals

poorly

represented

and only

cortex,

hippocampus

and septum.

a thin

and uniform

sheet

is

arranged

neurons

vesicles

(Kemali and Braintenberg,

~cetylcholine shown b y L o e w i

constituting

(ACh) i s (1937)

Cholineacetylase and the

highest

* To whom r e p r i n t

the

present

in

wall

1969). large

and by Cortell

was determined concentration

requests

of the

et

frog

concentrations

should

brain as

(1941).

b y Hebb a n d R a t k o v i c was f o u n d

simply

telencephalic

In the

al.

of

in the

be addressed.

(1964)

hindbrain,

Pharmacological Research Communications, Vol. 4, No. 2, 1972

136

particularly in the medulla and in the pons. A similar distribution was reported by Shen et al.

(1955) for

acetylcholinesterase. In mammals the highest concentrations in both cholineacetylase

(Hebb and Silver,

1956) and in ACh (Mc Intosh,

1941; Takahashi and Aprison, of the telencephalon,

1964) were found in some parts

namely the basal ganglia.

A number of drugs affect the level of ACh in mammal brain through different mechanisms of action (Pepeu,

1971). The

purpose of this research was to determine whether some of these drugs also affect ACh level in the less developed amphibian brain,

in which the cholinergic pathways seem to

have a completely different layout from that in the more developed mammalian brain.

MATERIALS AND METHODS

Frogs (rana esculenta),

20 -40 g

body weight~ were used. The drugs, g/l;

KC1 0 . 2 9

0.35

g/l),

lymphatic Thirty

dissolved in 0.2 ml of frog saline, g/l;

were

administered

sack.

Controls

minutes

after

by decapitation to

five

were

(1958).

within

48 h o u r s

muscle

according

the

extracts the

pooled

a

the

few minutes

solutionby

inactive bathing

the

of

facts: brought

in (a) to

the

dorsal

with

saline. killed

of

Three

ACh d e t e r m i n a t i o n , of

extracts

Smallman

the

dorsal

extracts

the

leech

pH 10 w i t h 0.1N

(1958).

w a s ACh w a s

extracts

with

and

was estimated

by Murnaghan

and neutralized both

each

used

NaHCO 3

the

were

method

the

g/l;

removed.

a preparation

present

were

frogs

for

by the

procedure

following

when they

were

of

in

in~ected

together

substance

by the

inactivated

to

0.7

was quickly

T h e ACh c o n t e n t

active

supported

boiled

brain

by means

glucose

only

injection

a n d ACh w a s e x t r a c t e d

Fisher

That

the

g/l;

by injection

were

and the

brains

weighed

CaC12 0 . 1 7

(NaC1 6.4

were 0.1NaOH, HC1;

(b)

the

when eserine

was omitted

leech

and when d-tubocurarine

muscle

from

Pharmaco~gicalResearch Communication~ VoL4, No. 2 , 1 9 ~

137

chloride

( 3 . 1 0 -6 g/ml) was a d d e d to the same solution.

recovery

of the ACh c h l o r i d e

a d d e d to the brains

The

was about

90%.

RESULTS

The e x p e r i m e n t s

of several

months,

evident was

during

the other

it was n e c e s s a r y between

treated

were made within The effect in Table in the

from J a n u a r y

that the level

significantly

of b r a i n

lower

carried

months,

and control

a period

and it became

ACh in the u n t r e a t e d May,

as shown

to c o m p a r e

out over

to September,

in April,

June and July t h a n

in Table

the levels groups

frogs

I. T h e r e f o r e

of b r a i n ACh only

in which

the d e t e r m i n a t i o n s

a week.

of a number

of drugs

2. P e n t o b a r b i t a l

frogs

increase

were

caused

at the dose

a 28% increase

in b r a i n ACh was

Brain ACh in the frog

in total

is r e p o r t e d deep a n a e s t h e s i a

b r a i n ACh.

following

An

the

1

in d i f f e r e n t

No. determinations

January February

inducing

also o b s e r v e d TABLE

Months

on A C h level

7

months.

ACh #g/g + S.E,

1 2 . 9 7 + 1. 28

March

April May June July

13

September

* This value

5

differs

significantly

8.96 + 0 . 3 7 *

11.55 ± 0.39

f r o m the others,

( P < 0.01)

Pharmacological Research Communications, VoL 4, No. 2, 1972

138

administration

of o x o t r e m o r i n e .

c a u s e d myosis,

increase

in the m u c o u s

secretions

skin,

decrease

in s p o n t a n e o u s

activity

a marked

the h i g h e s t However,

dose u s e d , t h e tremor

of s c o p o l a m i n e motility~ changes

in total

this

loss of the r i g h t i n g

was never

was f o l l o w e d

lasting

In the frog,

observed.

b r a i n ACh were

from the and,

at

reflex.

The a d m i n i s t r a t i o n

by an increase

10-15 m i n ~ a n d

drug

in the s p o n t a n e o u s

by mydriasis.

No s i g n i f i c a n t

found.

TABLE

2

L e v e l s of brain ACh in frogs t r e a t e d with d i f f e r e n t drugs. a n i m a l s w e r e k i l l e d 3 0 m i n after i n j e c t i o n into the dorsal l y m p h a t i c sack. Drugs

Dose mg/kg

No. experiments

ACh #g/g ~ S.E.

none

m

5

11.86

+ 0.30

Pentobarbital

50

5

15.22

+ 0.23

none

--

8

9.23

+ 0.53

4

9.15

+ 0.79

3

10.60

+ 0.56

2

10.50

1

Scopolamine none

m

Scopolamine

5

none

--

4

8.87

± 0.83

Scopolamine

10

4

10.13

+ 1.35

none

--

5

8.53

+ 0.44

The

Percent change

+28

0.01

n.s°

+14

n.s°

Oxotremorine

1

4

10.50 + 0.6

+23

0.05

Oxotremorine

2

3

11.30

+33

0.05

DISCUSSION that

The e x p e r i m e n t s

in the frog b r a i n

content, et al.

as p r e v i o u s l y (1941).

is about

there

of ACh found

higher

in t h i s

is a r e m a r k a b l y

shown by Loewi

The level

four t i m e s

reported

t h a n that

+ 0.97

paper

confirm

h i g h ACh

(1937)

and Cortell

in our

experiments

in the whole

rat brain

Pharmaco~gicalResearch Communications, ~ L ~ No. 2,1972 (Giarman and Pepeu~

139

1962) and three times higher than in

the caudate nucleus

of the cat (Pepeu,

1971). The ACh

content is very high both in invertebrates and in lower vertebrates tissues (Florey and Winesdorfer~

1968;

and Fessard~

1958). In the

1942; Augustinsson and Johnels~

brain of the octopus~

for instance~

220 ~g/g (Florey and Winesdorfer,

Feldberg

it ranges from 70 to

1968). An explanation

for the high ACh concentration in the frog brain could be offered by the complete lack of a white substance associated with the cortex ~n this animal species. cortical fibres~ too,

are unmyelinated

The intra-

(Kemali and Braitenberg~

1969). A comparison between the number of neurons per mm

3

determined by the latter authors in the frog brain and that reported by Tower and Elliott

(1952) for the

brain of several mammalians shows that the neuronal density in the frog cortex is almost 10 times higher than in the mouse and 30 times higher than in the cat cerebral cortex.

Tower and Elliott (1952) showed that in mammals

the ACh content in the brain decreases fairly regularly with ascending order in the phylogenetic scale.

The

decrease is related with that in the aYerage number of neurons per unit volume of the cortex. The possibility that the neurons in the frog brain are predominantly cholinergic in nature should also be considered. According to our results,

the level of brain ACh in the

frog undergoes a seasonal variation~

being lower in spring

and early summer than in the other months.

Information on

seasonal changes in the level of neurotransmitters~ in amphibians,

particularly

seems rather scarce. Seasonal variations were

observed in the ACh level of the motor and sensory nerves of the bull frog (Welsch et al.~

1971) and seasonal variations

in the sensitivity of peripheral nerves and C.N.S. of the frog to different neurotransmitters were reported (Cooper et al.,

Pharmaco~g~alResearch Commun~ations, VoL4, No. 2,1972

140

1970;

Curtis

system,

et

seasonal

also r e p o r t e d Bome

al.,

1961).

changes

(Welsch

remarks

In the

and Dettbarn,

can be made

species

by the drugs

induces

anaesthesia

and Lundgren, oxotremorine

tremor

Leslie

tremors

an i n c r e a s e

of o x o t r e m o r i n e et al.

observed

in mice

(Bartolini

1970)

in b r a i n ACh and sedation,

but no

following

in the rana

motility,

and Pepeu

(1964)

injection

pipiens

reminiscent

by

caused

an

of that

no change

in

s h o w e d that the d e c r e a s e of s c o p o l a m i n e

in the rat to the t e l e n c e p h a l o n . b r a i n ACh

et al.,

In the

in b r a i n ACh cause d by a d m i n i s t r a t i o n

does not change

(Holmstedt

in brain ACh.

and rats (Longo,1966)but

Giarman

Pentobarbital

as o b s e r v e d

In the frog s c o p o l a m i n e

in s p o n t a n e o u s

limited

ACh,

and a rise

w a s also n o t e d

increase

b r a i n ACh.

paper.

In the rat

The lack of t r e m o r s

(1969).

in some m a m m a l i a n

in b r a i n

and Pepeu, 1962).

causes

was seen.

exerted

and in the eat

system were

the effects

in the present

and a rise

1966)

frog we found

used

nervous

1970).

by c o m p a r i n g

in the frog with those

(Giarman

peripheral

in the c h o l i n e r g i c

exerted

in the rat

lobster

is

Scopolamine

in the c h i c k e n

(Pepeu,

unpublished

results). Centrally

acting

anticholinergic

of ACh from the cerebral Bartolini the

and Pepeu,

increase

cortex

1967).

in ACh output

and the d e c r e a s e

(Szerb

output

by a n t i c h o l i n e r g i c

cortical

lesions

a lesion

of the

primitive through level

fimbria

frog b r a i n

which

et al.,

in the cat

in m a m m a l s

A relationship

in the cortex caused

drugs

1970).

in the rat

(Szerb,

in A C h

is p r e v e n t e d

and Szerb, (Pepeu,

of ACh in c e r e b r a l

cortex.

drugs

1964;

in ACh content

1972).

could

Conversely,

by sub-

1969)

seems to lack the c h o l i n e r g i c

the a n t i c h o l i n e r g i c

the output

was found between

The i n c r e a s e

drugs

(Dudar

increase

and by

The more pathways

affect

the

a m u c h more

Pharmacological Research Communications, Vol. 4, No. 2, 1972

efficient synthesis of ACh

141

by the frog central neurons, as

compared to mammalian ones, could account for this marked difference.

This work was supported by grants Nos. 6902207 and 70.01045.04 from the Consiglio Nazionale delle Ricerche, Rome, Italy.

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