The effects of atrial natriuretic peptide on food-reinforced conditioning in rats. Interactions with neurotransmitters

Physiology&Behavior,Vol.53, pp. 325-328, 1993

0031-9384/93$6.00 + .00 Copyright© 1993PergamonPressLtd.

Printedin the USA.

The Effects of Atrial Natriuretic Peptide on Food-Reinforced Conditioning in Rats. Interactions with Neurotransmitters AMELIA BIDZSERANOVA,* JONY GUERON,* GABOR TOTHt

A N D G Y U L A T E L E G D Y *t

*Institute of Pathophysiology and ¢'Institute of Medical Chemistry, Albert Szent-GySrgyi Medical University, Szeged, POB 531, Hungary Received 21 J a n u a r y 1992 B1DZSERANOVA, A., J. GUERON, G. TOTH AND G. TELEGDY. The effects of atrial natriureticpeptide onfood-reinforced conditioning in rats. Interactions with neurotransmitters. PHYS1OL BEHAV 53(2) 325-328, 1993.--The effects of two doses of rat atrial natriuretic peptide (£ANP-1-28), 200 and 500 ng, on 6-day acquisition and extinction of food-reinforcedconditional learning (conditional stimulus: light) were studied in rats following administration into the lateral cerebroventricle. With the higher dose, there was a tendency for facilitatedacquisition and significantlydelayedextinction of the positivelyreinforcedlearning task. In order to clarifywhether the effect of the peptide is obtained through the involvementof neurotransmitters,the experimental animals were pretreated with different receptor blockers in selected doses that did not influence the behavioral test. Haloperidol, atropine, phenoxybenzamine, and propranolol blocked the action of ANP on extinction of the food-reinforced conditioning, whereas naloxone, bicuculline,and methysergidewere ineffective.The results suggestthat ANP might be considereda modulating agent in a positivelyreinforced conditional learning task, and that its action might involve dopaminergic, cholinergic,and a- and fl-adrenergic mechanisms. Atrial natriuretic peptide

Positivereinforcement

Conditional learning

THE presence of atrial natriuretic peptide (ANP) and its receptors in the central nervous system is well documented (6-1 I). Although the functions of this peptide in the brain are generally connected with water-salt homeostasis, the presence of ANP in motivation-emotion-relatedstructures (10) makes it reasonable to look for possible central effects of ANP on learning processes and memory formation. We earlier studied the effects of ANP on fear-motivated (negatively reinforced) learning in rats (1-4). The peptide facilitated consolidation of a passive avoidance response and delayed extinction of an active avoidance response in a dose-dependent manner. In the present study, the effect of ANP on food-reinforced (positively reinforced) conditional learning behavior in rats was investigated. To examine the involvement of neurotransmitters in the ANP-induced action, the animals were pretreated with different receptor blockers that had earlier been proven effective in modifying the action of a number of peptides in the same behavioral paradigm (13).

Behavior

Neurotransmitters

six per cage under standard conditions: constant room temperature, an artificial 12 h light- 12 h dark cycle (lights on at 0600) and free access to tap water. A 22-h food deprivation (+2-h feeding schedule) was initiated 3 days before the experiments. Behavioral sessions were carried out between 0800 and 1600.

Surgery The rats were anesthetized with pentobarbital-Na (Nembutal, 35 mg/kg) intraperitoneally and a 20 ga 1-1/2" Luer cannula was introduced into the right lateral cerebroventricle and fixed to the skull with dental cement (Spofa Cs). The stereotaxic coordinates of Fifkova and Marsala (5) were used (AP: +1.0, L: 1.5, V: 3.0). A volume of 2 #1 per animal was injected through the cannula, using a microinjector (Mauser, BRD) and tubing. The animals were used after a recovery period of 5 days. The correct positioning of the cannula was verified by injecting methylene blue after the experiments were completed. Data from animals with incorrectly positioned cannulae were excluded from statistical evaluation.

METHOD

Animals

Behavioral Method

Male rats of the CFY albino strain, weighing 180-250 g at the beginning of the experiments, were used. They were housed

The experimental apparatus was a conditioning box, measuring 12 X 20 x 40 cm, with a 2 X 2 cm slip-in feeding tray at

Requests for reprints should be addressed to Prof. G. Telegdy, Inst. of Pathophysiology, Medical University, Szeged, POB. 531, Hungary 6701.

325

326

BIDZSERAN()V,\ FI

.\I,

% 100"

ACQUISITION % (17) ANP 500 ncj (20) ANP 200 ng (20) Control

100 -

W U Z


.

I

I

i

!

i

i

2

3

4

5

6

one end. Above the tray, there was a 15 W bulb. At this end of the box, there was a 4 × 4 cm Plexiglas window for observation. The conditioning stimulus was light from the bulb for 10 s, and this was associated with food reinforcement of one oat grain in the feeding tray (12). Three animals were conditioned simultaneously in three adjacent boxes. On the fourth day of the deprivation schedule, 22-h food-deprived rats were placed in the conditioning boxes, with the feeding tray full of grain and the light on for 10 min. During this habituation period, all rats generally found the food and ate. On the following day, experimental sessions began. Each 10-min behavioral session consisted of 10 trials, with a mean inter'trial interval of 60 s (range 50-70 s). The conditional stimulus (light) and the food reinforcement were presented for a m a x i m u m of 15 s or until the rat picked up the oat grain from the tray. After each behavioral session, standard rat chow was given to the animals for 2 h. Conditioning was

+

ANP

"ii~---.~b..~.~..._""-~ (21) HoI.ANP (1"7) Control (23) Hal

I

I

I

i

i

1 2 3 4 5 p< 0.05 vs Control (M-W) ~ * p<0,01 vs Control (M-W) * p<0.01 vs Control (K-W)

doy

FIG. I. Effects of two doses of ANP on acquisition of food-reinforced conditioning in rats. The numbers in parentheses are the numbers of animals used.

-~-~

*~~(19)

:E rr 0 50b_ rr UJ

I

1

+

~.

UJ (J Z <

6

doy

FIG. 3. Effect of haloperidol (10 #g/kg IP) on ANP-induced action (500 ng/animal) on extinction of food-reinforced conditioning in rats. The numbers in parentheses are the numbers of animals used.

carried out for 6 days. The criterion of learning was eight or more conditioned feeding responses (CFR) during 3 consecutive days (80% or more performance). After the rats had reached the criterion of learning, extinction began. During extinction the conditional stimulus was not associated with food and the positive response was recorded. Extinction session was also carried out for 6 days.

Peptide Rat ANP (rANP-1-28) was purchased from Bachem. (Cal., USA) and also synthesized by G. T6th. The ANP obtained from two sources were chemically (HPLC) identical. The peptide was given in a volume of 2 ~1 ICV, in two doses, 200 and 500 ng/ rat, every day, 20 min prior to the conditioning or extinction trials.

EXTINCTION

%

% 1OO-

100. LIJ (J Z

•

n-

O b_ rr LU

_*

_ _

+

~ (12) ANP 500 ng

<

(16) ANP 200 ng

50"

(19) Control

i

i

i

i

i

1 2 3 4 5 * p<0.05 vs Control (M-W)

i

6

~" (19) ANP

o

50-

(17) Control (15) Atr (14) Atr,ANP

n, ILl

I

doy

.-x- p~O.01 vs Control (M-W) * p
FIG. 2. Effect of two doses of ANP on extinction of food-reinforced responding in rats. The numbers in parentheses are the numbers of animals used.

!

i

i

|

1 2 3 4 5 * p
!

6

doy

FIG. 4. Effect of atropine (2 mg/kg IP) on ANP-induced action (500 ng/ animal) on extinction of food-reinforced conditioning in rats. The numbers in parentheses are the numbers of animals used.

EFFECTS OF ANP ON FOOD-REINFORCED CONDITIONING

327

% 100-

e/o

100-

* (20) o

50-

rr LIJ n

ANP

~ (24) ANP

(20) (20)

Control Propr.

~

(20)

Propr.*ANP

uJ ra

09) Control (18) Phen ,ANP (17) Phen.

50-

I

I

I

I

I

I

1 2 3 4 5 * ~ p
I

I

I

I

I

I

6

1 2 3 4 5 , p<0.05 vs Control (M-W) ~ p
day

FIG. 5. Effect ofpropranolol ( l0 mg/kg IP) on ANP-induced action (500 ng/animal) on extinction of food-reinforced conditioning in rats. The numbers in parentheses are the numbers of animals used.

Experimental Groups In the first series of experiments, the animals were divided into three groups: a control group (which received 2 tA saline ICV) and two dose groups, which received 200 or 500 ng ANP per rat in a volume of 2 #1 ICV every day, 20 min prior to the acquisition or the extinction trials. In the following experiments, the animals were allocated into four groups: 1. Control groups: saline pretreatment (20-30 min prior saline, SC or IP) plus treatment with 2 ul saline ICV 20 rain prior to the trial. 2. Blocker-treated groups: receptor-blocker pretreatment (20 or 30 rain prior to saline SC or IP) plus treatment with 2/zl saline ICV) 20 rain prior to the trial. 3. Peptide-treated groups: saline pretreatment (20 or 30 rain prior to the peptide SC or IP) plus treatment with 200 or 500 ng ANP in a volume of 2 #1 ICV, 20 rain prior to the trial. 4. Blocker plus peptide-treated groups: blocker pretreatment plus treatment with ANP.

Statistical Analysis Statistical evaluation of the data was performed with the nonparametric tests ofKruskal-Wallis (KW) followed by MannWhitney (MW). A probability level of 0.05 or less was considered significant.

day

FIG. 6. Effectof phenoxybenzamine (2 mg/kg IP) on ANP-induced action (500 ng/animal) on extinction of food-reinforced conditioning in rats. The numbers in parentheses are the numbers of animals used.

Pretreatment Pretreatment was performed with the following receptor blockers: haloperidol (G. Richter, Budapest) (10 /~g/kg, IP), atropine (EGIS, Budapest) (2 mg/kg, IP), propranolol (I.C.I. Ltd., GB) (10 mg/kg, IP), phenoxybenzamine (Smith, Kline and French, GB) (2 mg/kg, IP), methysergide (Sandoz) (5 mg/kg, IP). All the above-mentioned drugs were given 30 min before the peptide. Naloxone (Endo Lab. Inc.) (0.3 mg/kg, SC) and bicuculline (Serva) ( 1 mg/kg, SC) were given 20 min before the peptide. The treatment was carried out only before the extinction trial.

6

RESULTS

The doses of 200 and 500 ng/rat of ANP were selected on the basis of previous experience (1-4). As shown in Fig. 1, with the higher dose there was a tendency for more rapid acquisition of the food-reinforced conditioning, but this did not reach statistical significance. In Fig. 2, the effects of these two doses of ANP on extinction of the conditioning can be seen. The dose of 500 ng/rat significantly delayed extinction of the conditioned response during all 6 days of testing (KW and MW tests at p < 0.001). The lower dose was ineffective. In the receptor blocker combined study, only 500 ng/rat ANP was used. When the animals were pretreated with different receptor blockers in appropriate doses, the following results were obtained: haloperidol (Fig. 3) in a dose of l0 #g/kg IP, administered 30 min before the peptide every day, significantly blocked the delaying effect of ANP on extinction of the conditioned response

% 100. ÷

*

(15) ANP (12) ANP *Bicuc

LIJ (J Z

:E

nO

LL rr LIJ Q.

(16) Control (12) Bicuculline

50-

I

/

i

i

i

i

1

2

3

4

5

6

}i p< 0,05 vs Contr

~

doy

(M-W)

p(O01 vs Contr (M-W~ , p, O05 vs Contr (K-W)

FIG. 7. Effect of bicuculline (1 mg/kg SC) on ANP-induced action (500 ng/animal) on extinction of food-reinforced conditioning in rats. The numbers in parentheses are the numbers of animals used.

328

BII)ZSERAN~)~:\ t7-1 . \ 1

on most days (on second, third, and tburth days, p < (I.05. MW. and on the fifth and sixth days, p < 0.001. K W and MW). In a similar way, atropine, phenoxybenzamine, and propranolol (Figs. 4, 5, and 6) modulated the effect of the peptide. The other three receptor blockers (naloxone, bicucultine, and methysergide) were ineffective (only the results obtained with bicuculline are shown in Fig. 7). DISCUSSION The present study demonstrates that ANP is effective in a positively reinforced learning task. When administered ICV in a dose of 500 ng/rat, 20 min prior to the daily acquisition or extinction sessions, it transitorily enhanced acquisition, and significantly delayed extinction of the food-reinforced conditioning during all 6 days on which extinction of the conditional response was measured. These results confirm our previous observations that ANP might be considered a neuropeptide with a regulatory function in learning processes, because in some fear-motivated learning tests (negatively reinforced learning) the peptide successfully facilitated consolidation of passive avoidance learning and delayed extinction of active avoidance learning (1-4). As we have shown in our previous negatively reinforced experiments that ANP lengthened the passive avoidance response, delayed the extinction of active avoidance conditioning, and

ANP cannot have an afl~ct on locomotion in 24 h test (4), made our position strong enough to suppose that ANP has an efl~ct on learning and memory, function (1-3). The present paper is a continuation of the previous experiments demonstrating that the extinction of a positively reinforced conditioning can also be delayed. O f course, one has to be aware of the fact that the delayed extinction alone cannot be interpreted as an index of memory formation. Because food intake cannot be influenced by ANP (6), the motivation for food intake--hunger drive-can be ruled out. Taking together, the results of the negatively reinforced experiments with the presenl one. we feel that thc evidences altogether are strong enough to suppose that ANP acting on learning and memory formation connected with positively reinforcement of the conditioning also. Regarding the transmitters involved in the positively reinforced conditioning, it seems that, in contrast to the negatively reinforced conditioning, in which dopaminergic and cholinergic transmitters were involved here, not only dopaminergic and cholinergic but ~- and/3-adrenergic neurotransmitters may be involved also. This will give an important difference in organization of between the negatively and positively reinforced learning processes modulated by ANP. ACKNOWLE[X~EM[!NfS The work has been supported by the Ministry, of Social and Welfare of Hungary (T-70/1990) and OTKA (T/3, 1354).

REFERENCES 1. Bidzseranova, A.; Telegdy, G.; Penke, B. The effects of atrial antriuretic peptide on passive avoidance behavior in rats. Brain Res. Bull. 26:177-180; 1991. 2. Bidzseranova, A.; Telegdy, G.; T6th, G. The effects of receptor blockers on atrial natriuretie peptide-induced action on passiveavoidance behavior in rats. Pharmacol. Biochem. Behav. 40:237-239; 1991. 3. Bidzseranova, A.; Gueron, J.; Penke, B.; Telegdy, G. The effects of atrial natriuretic peptide on active avoidance behavior in rats. The role of the transmitter systems. Pharmacol. Biochem. Behav. 40(1): 61-64; 1991. 4. Bidzseranova, A.; T6th, G.; Telegdy, G. The effects of atrial natriuretic peptide on the open-field activity of rats. The role of neurotransmitters. Neuropeptides 20:163-167; 1991. 5. Fifkova, E.; Marsala, J. Stereotaxic atlases for the cat, rabbit and rat. In: Bures, J.; Petr~n, M.; Zachar, J., eds. Electrophysiological methods in biological research. Prague: Publishing House of the Czechoslovak Academy of Sciences; 1967:653-731. 6. lnagami, T.; Tanaka, I. K.; McKezie, J. C.; Nakamura, M.: Takayanagi, R.; Imada, T.; Pochet, R.; Resibois, A.; Naruse, M.; Naruse, K.; Shibasaki, T. Discovery of atrial natriuretic factor in the brain: Its characterization and cardiovascular implication. Cell. Mol. Neurobiol. 9(1):75-85; 1989. 7. Kawata, M. K.; Nakao, K.; Morii, N.; Kiso, J.; Jamashita, H.; lmura, H.; Sano, J. Atrial natriuretic polypeptide: Topographical distribution

8.

9. t0. 11.

12. 13. 14.

in the rat brain by radioimmunoassay and immunohistochemistry. Neuroscience 16:521-546; 1985. Morii, N.; Nakao, K.: Sugawara, A.; Sakamoto, M.; Suda, M.; Shimokura, M.; Kiso, J.; Jamori, J.; Imura, H. Occurence of atrial natriuretic polypeptide in brain. Biochem. Biophys. Res. Commun. 127:413-419; 1985. Quirion, R.; Dalpe, H.; De Lean, A.; Gutkowska, J.; Cantin, M.; Genest, J. Atrial natriuretic factor (ANF) binding sites in brain and related structures. Peptides 5:1167-1172; 1984. Skofitsch, G.; Jacobowitz, D. Atrial natriuretic peptide in the central nervous system of the rat. Cell Mol. Neurobiol. 4:339391; 1988. Tanaka, I.; Misono, K. S.; Inagami, T. Atrial natriuretic factor in rat hypothalamus, atria and plasma: Determination of specific radioimmunoassay. Biochem. Biophys. Res. Commun. 124:663-668; 1984. Telegdy, G.; Hadnagy, J.; Lissfik, K. The effect of gonads on conditioned avoidance behavior of rats. Acta Physiol. Acad. Sci. Hung. 37:439-444; 1968. Telegdy, G. Neuropeptides and brain function. Basel: Karger; 1987: 1-332. Zamir, N.; Skofitsch, G.; Eskay, R. L." Jacobowitz, D. M. Distribution of immunoreactive atrial natriuretic peptides in the central nervous system of the rat. Brain Res. 365:105-111: 1986.