ISSN 0012�4966, Doklady Biological Sciences, 2014, Vol. 454, pp. 16–18. © Pleiades Publishing, Ltd., 2014.Original Russian Text © A.N. Inozemtsev, D.S. Berezhnoy, T.N. Fedorova, S.L. Stvolinsky, 2014, published in Doklady Akademii Nauk, 2014, Vol. 454, No. 5, pp. 606–608.

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Carnosine (β�alanyl�L�histidine) was firstdescribed by Russian biochemists in the muscle tissue[1]. In humans and other mammals, carnosine andsimilar compounds are observed in large quantities inthe muscles, heart, and some brain regions, such as theolfactory bulb. Carnosine has some properties, includ�ing antioxidant and membrane�protective activities,playing an important role in elevation of stress resis�tance [2, 3].

These properties are important because oxidativestress is involved in the pathogenesis of neurodegener�ative pathologies, including Alzheimer’s disease, Par�kinson’s disease, ischemia, multiple sclerosis, andothers [4–7].

Oxidative stress results in development of func�tional and structural impairments in various biologicalsubstrates of the brain tissue. Polyunsaturated fattyacids, the principal constituents of cell membranes,are very sensitive to the damaging effects of active oxy�gen species due to a low activity of the system of anti�oxidant defense in the brain and the presence of tran�sition metal ions. In turn, the products formed in reac�tions of lipid peroxidation are highly toxic and mayinduce cell death. Exhaustion of the system of antiox�idant defense promotes the development of oxidativestress; therefore, application of antioxidants may benecessary under these conditions.

In different experimental models of CNS diseases,such as acute hypobaric hypoxia, global and focalischemia, and Parkinsonism, carnosine has beenshown to prevent the development of oxidative dam�age to the brain tissue and to improve the efficacy ofthe endogenous system of defense against oxidativestress [5, 8]. In the experimental model of prenatalhyperhomocysteinemia in rats, which is associatedwith impairments of cognitive functions, addition of

carnosine to the animal diet significantly improveslearning and memory processes [9].

Administration of carnosine to animals subjectedto damaging factors decreased mortality, improvedphysiological parameters and neurological state, andpromoted preservation of skills acquired prior toischemic brain damage [10].

Here, we studied the effects of carnosine on learn�ing and memory of animals trained in a task with neg�ative reinforcement with an electric pain stress.

Thirty�three white rats weighing 250–300 g wereused in the study. The animals were trained to performactive avoidance in a shuttle box. Training consisted of8 sessions with 20 presentations of stimuli performedevery other day. An experimental chamber wasequipped with an electrifiable floor and divided intotwo equal compartments with a partition with a hole.The following protocol was used for the training: aconditioned stimulus (10�s light) was presented in thecompartment where the animal was; 10 s later, anunconditioned stimulus (10�s electrical current) wasapplied. If the animal moved to the opposite compart�ment of the chamber, then the stimuli were turned offand a 30�s intertrial interval was made. Movement ofthe animal to the other compartment in response tothe conditioned stimulus was considered a condi�tioned avoidance response. We recorded avoidanceresponses, escape responses, i.e., moving after the footshock, and intertrial crossings, i.e., moving during theintertrial interval.

The animals of the experimental group were intra�peritoneally injected with carnosine 100 mg/kg 1 hprior to each experiment (n = 17). The control ani�mals (n = 16) were injected with an equal volume ofphysiological saline.

We considered the number of avoidance responsesexpressed as a percentage of the total number of pre�sentations in the experimental session to be the mainindex of learning. When the animal exhibited avoid�ance response in 75% of recall trials in the session, we

The Effect of the Natural Dipeptide Carnosine on Learning of Rats under the Conditions of Negative Reinforcement

A. N. Inozemtsev, D. S. Berezhnoy, T. N. Fedorova, and S. L. StvolinskyPresented by Academician N. F. Myasoedov April 19, 2013

Received April 25, 2013

DOI: 10.1134/S0012496614010177

Moscow State University, Moscow, 119992 Russia

PHYSIOLOGY

DOKLADY BIOLOGICAL SCIENCES Vol. 454 2014

THE EFFECT OF THE NATURAL DIPEPTIDE CARNOSINE 17

regarded this as stable learning; learning was consid�ered unstable when the animal exhibited 50–75%avoidance responses; the animals with less than 50% ofcorrect responses were considered unlearned. We alsoused additional indices of animal learning and mem�ory. The avoidance responses exhibited in the first orsecond trial of each next session were considered to betraces of memory formed in the preceding session. Wealso recorded triple sequences of avoidance responsesin each session. Meeting these criteria is indicated byindicators in the figures.

Data in the figures and text are presented as themean and standard error of the mean. Statistical anal�ysis was performed using Fisher’s test for variances,Pearson’s goodness�of�fit test for comparison of indi�ces in the experimental and control groups, and theMann–Whitney test for comparison of differences intwo independent samples.

Our data show the accelerating effect of carnosineon learning, which was expressed in the time course ofelaboration of the avoidance response (Fig. 1). Thenumber of avoidance responses was higher than in thecontrol after experimental session 4 and significantlyhigher in sessions 5 and 8. An increase in the numberof avoidances observed in session 5 was preceded by anelevation of group variance, which was significantlyhigher in session 2 compared to the control (Fig. 2).Taking into account our previous data on elevatedvariance after treatment with learning enhancingdrugs [11], we suppose that the increased behavioral

heterogeneity after carnosine treatment may beregarded as a factor reflecting its positive effect.

Detailed analysis of training process revealed somedifferences between the groups even in session 1. Thecarnosine�treated animals were more active upon thefirst exposure to the experimental conditions and per�formed 7.9 ± 0.9 intertrial crossings versus 4.5 ± 0.5crossings observed in the control animals (p = 0.0024);the first avoidance response was observed in 94% of thecarnosine�treated rats versus 65% in the control group(p = 0.008). According to our previous data, anincrease in the number of intertrial crossings at theearly stage of training correlates with the level ofavoidance responses [12].

In the first and second trials of experimental ses�sion 3, the avoidance responses were observed in thecarnosine�treated and control animals, which weregard as recall of memory traces formed in the previ�ous sessions (Fig. 1, see indicators). Reproduction ofthe avoidance response in three consecutive trialsappeared simultaneously in the animals of both groupsduring session 4 (Fig. 1, see indicators). The differ�ences between the groups were observed in stablereproduction of the avoidance response at the level of75% in session 5, when 41 and 6% of the carnosine�treated and control animals, respectively, met the cri�terion (Z = 2.75, p = 0.006).

At the end of training, 33% of the control animalsreached the 75% level of stable avoidance performance(Fig. 3), whereas 65% of the carnosine�treated animalsexhibited a similar level of performance (p = 0.02).

We can conclude that carnosine improved learningbased on application of electrical pain stress. At theearly stage of training, carnosine treatment resulted in

100

80

60

40

20

0

Avoidance responses, %

1 2 3 4 5 6 7 8Days

Fig. 1. The time course of active avoidance training in ashuttle�box in the experimental animals. Data are pre�sented as M ± m. Here and in Fig. 2, abscissa axis, consec�utive days of training. Ordinate axis, avoidance responses,% of total number of trials. Black line, control group;dashed line, carnosine�treated group. Circle on therespective line, appearance of the avoidance response inthe first and second trials; square on the respective line,appearance of the triple consecutive avoidance response.(*) p < 0.05 according to the Mann–Whitney test.

30

0

Variance of avoidance responses, %

1 2 3 4 5 6 7 8Days

20

10

Fig. 2. Group variances of the number of avoidanceresponses in the groups of experimental animals duringtraining. Ordinate axis, variances of the number of avoid�ance responses. Black line, control group; dashed line,carnosine�treated group. (*) p < 0.05 and (�) p < 0.08according to Fisher’s test.

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INOZEMTSEV et al.

an increase in the number of intertrial crossings, indi�cating both an enhancement of orienting�exploratoryactivity and an acceleration of appearance of the firstavoidance response. The beneficial effect of carnosinewas also observed in the formation of stable recall ofthe avoidance response at the level of 75%.

The main physiological effect of carnosine isrelated to its antioxidant properties [10, 13]; therefore,we can suppose that the activating influence of carnos�ine is related to its protective effect on the nervous tis�sue under the conditions of electrical pain stress. Thisis specifically important in the first session because ofmore frequent foot shocks. The total buffering actionof carnosine and normalization of metabolism [14]may improve the functional capabilities of the nervoustissue under these conditions. It is supposed that car�nosine may modulate the hypothalamic–pituitary sys�tem, alleviating the stress response [15]. Moreover, ourdata on stabilization of performance of the avoidanceresponses after carnosine treatment also allow us tosuggest the stabilizing effect of the drug on the func�tional state of animals formed during training.

Thus, our data demonstrate that carnosineimproves learning due to acceleration of the appear�ance of the avoidance response at the initial stage oftraining and results in the higher level of performanceat the end of training.

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Translated by M. Stepanichev

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0CarnosineControl

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*

*

Unlearned

Unstable recall

Stablerecall

%

Fig. 3. Training in a shuttle�box in the experimental animals in session 8. Ordinate axis, number of animals, percentage of thetotal number of animals. Undashed area, animals exhibiting 75% level of avoidance responses in the session; diagonal ruling, ani�mals with 50–75% probability of avoidance responses in the session; gray area, unlearned animals. (*) p < 0.05 according to Pear�son’s goodness�of�fit test.