effects of po3tnatal influences on conditioning in rats · 2012. 9. 26. · effects of postnatal...

ACTA NEUROBIOL. EXP. 1974, 34: 99-111

Lecture delivered a t Symposium "Brain and Behavior" held In Jablonna near Warszawa

July 1972

EFFECTS OF PO3TNATAL INFLUENCES ON CONDITIONING AND SOME FUNCTIONAL AND STRUCTURAL BRAIN PARAMETERS

IN RATS

J. MYSLIVWEK, J. SAFAN'DA, J. HASMANNOVA, Z. OHALOUPICA and B. SEMIGINOVSKP

Department of Pathophysiology, Faculty of Medicine, Charles Univer8ity Plzefi, Czechoslovakia

Abstract. Early postnatal influences were studied in rats in three dfferent ways: (I) different litter size, (ii) feeding with restnicted amonn't of protein, (iii) different level of afferent stimulation. A complex evaluation of spontaneous activity, conditioning, electrophysiological and biochemical analyses was used in these experiments. Medium sized litters showed the greater electrophysiological reactivity (cortical evoked potentials) and the most stable performance in different kinds of conditioning (avoidance) experiments. Animals reared on a low protein diet were subnormal in electrophysiolog~cal indices, and had a lower level of biochemical activity for materials Involved in neural excitability, eleclrogenesis, and cellular and subcellular energetic metabolism (phospholipids, proteins, DNA, RNA). In all respects (behavioral, electrophysiological and biochemical tests) animals with partial sensory deprivation in the pastnatal period had the lowest indices. The "stimulated" animals on the other hand were superior to both other groups i n the tests used for the analysis of higher nervous activity, but electrophysiological and biochemical analyws did not show clear differences between the "sti~nulated" and control animals. Optimal developmenlt of brain functions requires adequate conditions in the early postnatal period. Hiowaver the "adequacy" of them conditions has d i l l to be determined more precisely.

The importance of early periods of life for the further development of the central nervous system is generally recognized. Two principal groups of early influences may be distinguished - nutritional factors

100 J. MYSLIVECEK ET AL.

and afferent influences (in spite of the fact that both are acting in per- manent mutual dependence).

Some attention has been given to these problems even in the past century (e.g., 5, 8, 24, 25) but rwently they have been studied more systematically (e.g., 1, 2, 6, 7, 9, 11, 21, 27). Our laboratory contributed to these studies by a complex approach, chiefly by means of electrophysiological analysis of the sequelae of early influences (3, 4, 10, 12-20, 23, 24, 26, 28).

The present work involves three different experimental designs: (i) unequal litter size, (ii) rearing on a low protein diet, and (iii) increased or decreased level of afferent stimuli in the early postnatal period.

METHOD

Wistar rats of both sexes were used in the experiments 1. Offspring of unequal litters were divided in three groups to make

litters of: A, 3-4, B, 7-8 and C, 13-15 pups. 2. Low protein diet (5 cal 010 of protein, 17 cal 010 of lipids and

78 cal 010 of carbohydrates) was administered to lactating females and after weaning at the age of 21 days it was given to the young until the 8th week of life.

3. Increased level of afferentation (Group SB) was achieved by complex stimulation 3 times a day for half an hour (from the 5th to 35th day) with light and sound stimuli (3 sec intervals) combined with som- esthetic-kinaesthetic stimulation (rotating cylinder with circumferential velocity 200 mmlmin) whereas the animals with a decreased level of afferent stimulation - partially deprived (Group DB) were reared and kept in darkness up to 35th day with minimal stimulation connected with cleaning. All animals in these experiments were offspring of medium sized litters and thus control animals were marked as in the first series - Group B.

Spontaneous exploratory activity was measured in a compartment with automatical registration of horizontal and vertical movements; conditioning was tested by an apparatus with semiautomatic or automatic programming which elaborated the avoidance reaction. Averaged cortical evoked potentials to sound and light stimuli were recorded in animals immobilized with succinylcholine-iodide. Biochemical analysis of the cere- brum was carried out after weighing, homogenization and determination of the dry weight; cholesterol, phospholipids, DNA, RNA, protein cholinesterase activity, sodium and potassium contents, were determined. A summary of the procedures used in experimental groups is given in Table I.

EFFECTS OF POSTNATAL INFLUENCES ON CONDITIONING 101

TABLE I

Summary of procedures

- - - - -- - - -

Different litter size 1- + I + L - - -

Low protein + I - , - I

- -- - - -- -

Level of sensory input + 1 + I 1 I - A- - ----- -

-- - \

\ Measurement 1

I Exploratory Avoidan%

RESULTS

- -- - -- - - I

Evoked Biochemical potentials 1 analysis

I

Different litter size

Animals of Group C (large litters) showed significantly higher exploratory activity (horizontal component) and also a more intense emotional reaction (def,ecation) to nociceptive stimulation than animals of both Groups A and B. In the three experiments with different schedules of avoidance conditioning, there were practically no significant differences. However, in the second series a break of 2 weeks caused animals of Group A (small litter) to show a statistically significantly lower number of avoidance reactions than animals of either other groups (evaluated in five experimental session before and after the break) (Fig. 1). Extinction of avoidance in the third experiment was significantly slower in Group C than in Groups A and B, which may be related to their higher reaction to nociceptive stimuli (18, Fig. 1).

B R E A K 1 7,

Fig. 1. Mean values of avoidance reactions (AV) in three groups of rats i n different sized litters (A. B. a n d C ) calculated born five sessions julst before and after a break i n the experiment of 14 days (10 trials per session). Significant diflerences indicated by arrows

:P < 0.05).


Cortical responses to sound (clicks) and light (flashes) at different stimulation rates were characterized in Group B by the shortest latencies and the highest amplitudes (Fig. 2 and 3). Animals from small a s well as large litters had longer latencies and lower amplitudes. The latency differences are significant however in some components within stimulation frequencies 0.21sec and llsec only (Table 11). The i.v. administration of pentobarbital, accentuates the differences betw,een groups without chang- ing their basic relations.

10 20 30 40 50 60 Mrnsec 10 20 30 40 50 60 Mmsec

Fig. 2. Mean values of cortical evovked potentials (EP,s) in the temporal cortex to auditory stimuli at different f~requencies. The same groups of animals as in Fig. 1. Abscissae, latent periods in msec; ordinates, amplitudes (arbitrary scale, true cali-

bration given on right, standard errors of means indicated).

Low protein diets (LP)

In this series of experiments only females were wed. The experiments were performed at age 15-16 weeks. The exploratory activity of the animals was measured in a single session of 10 min, which revealed that the LP animals were significantly more active in vertical activity (p < 0.05) than the controls without significant differences in the horizontal component.

EFFECTS OF POSTNATAL INFLUENCES ON CONDITIONING

vis

Fig. 3. Mean values of cortical E P s to visual s t imul i i n the occipital cortex. Other details as in Fig. 2.

TABLE I1

Significance of differences between mean latency values (to peak) of components PI and N1 of evoked potentials (EP) to auditory and visual stimuli in appropriate cortical areas (by Student's

t-test)

Visual non sign. ' non sign. 0.02 0.05 non sign. I non sign. -

Stimuli

Auditory 0.2/sec non sign. 1 0.05 non sign. non sign.

Comparison of groups I

1 l/sec / non sign. 1 non sign. non sign. 1 non slgn. - - - - - - --

The next day, average cortical EPs were recorded and the brains were then biochemically analped. The auditory cortex of LP animals were less reactive (longer latencies, lower amplitudes) to clicks (Fig. 4).

Modality

B I A I B C Frequency i EP component 1 - - - - - - ~ o m p ~ ~ - - p

- - - - - .- -- - - I


The evidence of significant differences only within lower frequency ranges is due to the fact that for the mean values the responses were counted only in animals in which the corresponding component was easily detect-

AUD-T

+* Fig. 4. Mean values of cortical EPs to auditory stimuli in the temporal cortex in rats on low protein (LP) diet and in contra1 fe-

W S ~ C sopv males (N). Significant differences of latencies

25 mrnc and amplitudes indicated.

able, and thus animals not responding within higher frequency ranges were eliminated in the evaluation of higher stimulus frequencies. The differences in the visual cortex were less evident and the absence of a response to a higher stimulation rate in some LP animals caused ap- parently better results at higher frequencies in the LP group (Fig. 5). The percentage of animals not responding at higher frequencies to stimuli of either modality was about 50010 in the LIP group whereas about 10010 in control.

There was s'ignificant decrease of the total weight and of total contents of DNA, RNA, proteins, phoispholipids and cholinesterase activity in the brains of the LP rats (Fig. 6). The comparison of the concentrations calculated from the obtained values showed a decrease of DNA and RNA concentrations and a small increase of cholesterol and protein. The con- centration ratios phospholipid/protein, phospholipid/cholesterol and K+/ /Na+ were significantly lower in the LP group. Thus it may be seen that protein deficiency caused a decrease of the brain cell number and evidently a deterioration of the subcellular energy processes requiring nor-


ma1 phospholipid metabolism. It was interesting that individual animals with inferior electrophysiological parameters, which were unable to fol-

Fig. 5. Same as in Fig. 4 for EPs to visual stimuli in the occipital cortex.

low a higher stimulation rate, were characterized also by low DNA and phospholipid contents, acetylcholinesterase activity and a K+/Na+ ratio.

Level of afferent stimulation

Spontaneous exploratory activity wals measured in a semi-darkened room in four sessions (four successive days) at the age of 6 weeks. Rats of the Group SB were most active in the vertical as well as in horizontal' components. and did not habituate from day to day. The lowest activity in the DS group was significantly different in the vertical component only (if compared with the controls).

The avoidance reaction was elaborated in semi-darkness until a criterion of three succesive avoidances in three succesive days was reached. Animals reared in darkness needed 44 trialk, control animals, 34 trials. Stimulated animals required 28 trials cmly (Fig. 7). A similar criterion for extinction of the avoidance reaction was reached in SB animals after 38 trials. in the control Group B after 50 trials, whereas DB rats needed 72 trials. The differences between the Group DB and both other groups were highly significant.


Iwelghf * ( DNA* ( R N A * I proteio*lphospho~* I Fig. 6 . Differences of mean values of brain weight and composition, cholinesterase activity and potassiumlsodium ratio between rconbmls (white columns) and LP anl- mals (black columns). Per cent scale, for all estimations, relative to control group; different m l a s for absolute measures, according to unit used in the estimations.

Standard deviations indicated; asterisk, significant differences ( p < 0.01).

/ l Y O i O A N I l 60 E L A B O R A T I O N

Fig. 7. Number of trials to criterion (ordbnates) of avoidance e~stablishing and its extinction (see text) in three groups of rats with different levels of stimulation in the early postnatal period. Standard errors of means indi-

ca,ted.

The cortical potentials evoked by acoustic stimuli in the auditoly cortex in the Group DB show lower amplitudes and longer latencies, mainly if compared with those in SB animals (Fig. 8). In the visual cortex the optimal parameters of potentiars evoked by flashes were recorded in the Group B (Fig. 9).


Biochemical analyses did not reveal significant (Fig. 10) differences between B and SB groups. The DB animals showed significantly lower values of many important components if compared to the control animals

A U D - T ----- B

Fig. 8. Mean values of mrtical EPs to 8/sec

auditory stimuli in the temporal tor-

tex of rats with different level of early stimulation. Other details as in Fig. 4. 2 5 m w

(Group B) and in some cases also when compared to SB animals (in which the results were more variable). If these data are related to the DNA content, i.e. to cell number, it may be seen that the cholinesterase activity per cell is significantly higher in the deprived animals than in both other groups (both p < 0.01). Their ganglioside contents are also higher than in the Group SB (p < 0.05). The phosphatidylserine per cell ratio in Group DB was significantly lower than in the control (B) animals.

DISCUSSION

The first series of experiments revealed in Group B (medium sized litters) the best ability to follow external stimuli with an electrical

108 J. MYSLNECEK ET AL.

response in the specific projection areas. In conditioning, animals of this group did not show any signs of a deteriorated reaction as in Group A after a break in the experiments, or in Group C - poorer extinction.

Fig. 9. Same as Fig. 8 for

I 1 c--.

to visual stimuli in the occipital I 2s rnsec cortex.

Moreover, animals of this group were better in some indices of diffe- rentiation (even if not statistically significant). Thus the animals of medium sized litters which have evidently the most adequate nutrition, as well as an adequate level d stimuli in the nest, showed the best parameters in the studied functions.

In animals reared ion a low protein diet we obtained with our electrophysiological and biochemical estimations an indirect evidence - for the time being- that their subnormal development of structures, and of mechanisms involved in excitation and electrogenesis is among the most important causes of their generally impaired dynamics of neural function.

The indices of higher nervous activity - elaboration of a conditioned avoidance reaction and its extinction - showed the following sequence of increasing level of performance: partially deprived anirnals<controls<stimulated animals. However the bioelectrical and biochemical

INDEX 1

I 4MOl I M I N

Fig. 10, Differences of mean values of brain weight and compssition in three groups of animals with different levels of afferent stimulation in the early postnatal period. White columns, partially deprived animals (DB); hatched c o l w , contlwls (R): blaclc columns, stimulated animals (SR). Two asterisks, differences significant a t the 5Oiu level. Other details

as in Fil:. 6.

I 1 ghorybel ip. ;bs@osrnlt w e i g h t c h o l e s t e r o l R N A DNA p r o t e i n c e r e b r o s i i i r n g l i o s i d C H E


analyses showed only clear and significantly decreased values in the deprived group. The stimulated group was less homogenous, and thus we can only suggest that the applied amount of stimulation was optimal for some individuals, whereas for others it exceeded their capacity and was stressful in nature. This view requires, however, further experimental analysis and the really adequate level of stimulation has to be determined.

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Received 3 July 1972

Jaromir MYSLIVECEK, Institute of Hygiene and Epidemiology, Srobhrova 48, Prague 10, Czechoslovakia, 10042.

Jifi SAFANDA, Zdenek CHALOUPKA and Bohdan SEMIGINOVSKP, Department of Patho- physiology, Faculty of Medicine, Charles University, Plzefi, Czechoslovakia.

Jarmila HASSMANNOVA, Department of Physiology, Medical Faculty of Hygiene, Ke Karlovu 4, Prague 2, Czechoslovakia.

effects of po3tnatal influences on conditioning in rats · 2012. 9. 26. · effects of postnatal...

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