Psychomotor Functions in Developing Rats ... - Science Direct

Neuroscienceand BiobehavioralReviews,Vol. 19, No. 3, pp. 413-425, 1995 Copyright ©1995ElsevierScienceLtd Printed in the USA.All rights reserved 0149-7634/95 $9.50 + .00

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Psychomotor Functions in Developing Rats: Ontogenetic Approach to Structure-Function Relationships ABDOULAYE

B,A ~ A N D B I A L L I V. S E R I

D~partement de Biologie et Physiologie Animale, Facult~ des Sciences et Techniques, Universitd Nationale, 22 B.P. 582 Abidjan 22, C6te d'lvoire R e c e i v e d 16 D e c e m b e r 1993 BA., A., AND B. V. SERI Psychomotorfunctions in developing rats: Ontogenetic approach to structure-function relationships. NEUROSCI BIOBEHAV REV 19(3) 413-425, 1995.-The functional development of the central nervous system (CNS) in the rat was studied from the 10th to the 45th postnatal day, through the ontogeny of psychomotor and sensory functions, by a battery of behavioral tests. The ontogenetic development of 10 different functions was described. The results showed that novelty-induced functions matured progressively in an adult-like pattern of functioning in the 3rd postnatal week. Indeed, exploratory activity was low at the 10th day, increased significantly to reach highest values from 20th to 30th postnatal day, then declined at the 45th postnatal day. No habituation was exhibited by 10- and 15-day-old rats; it appeared at the age of 20 days. Emotional reactivity induced by the novelty of surroundings clearly appeared from the 20th postnatal day, when the features of adult animal were reached. It appeared also that reflex and automatic motor functions came to maturity by the age of 3 wk, while voluntary motor functions continued to improve until the 30th day. Thus, the latency of the hind paw lifting reflex occurrence significantly decreased from the 10th to the 20th postnatal day, when the most improved values were reached. The wire-grasping times increased from the 10th to the 25th postnatal day in an exponential fashion. Locomotor activity developed significantly from the 10th to the 15th day, when the mature locomotion pattern was exhibited. The coordination of complex movements and motor initiative appeared only after the 20th postnatal day. The latencies of execution of crawling along the wire and of leap onto the ground decreased significantly from the 20th to the 45th day. These studies reveal the presence of the caudal to rostrai sequence of CNS development, predicting a spatio-temporal functional maturation of nuclei and centers in the rat's CNS. The building of the time-sequence of regional maturation of the brain integrated activities was attempted. Developing rats Brain ontogeny Novelty-induced functions Structure-function relationships Ontogenetic mechanisms

INTRODUCTION

Motor functions

tional sequence (38). For example, regional studies of DNA content quantifying cell number as a function o f age indicated that in the cerebrum it increases at slow rate until Day 21, increases at more rapid rate in cerebellum stopping at Day 17, and in the brainstem levels off on Day 14 (30). According to Balasz et al. (5), the amount of DNA doubles in the cerebrum and increases 35 times in the cerebellum from birth until weaning. The life history of individual neurons is also characterized by a temporal progression of developmental steps which include proliferation, migration, differentiation, formation of axonal pathways and synaptic connections, and the onset of the physiological function (38). The development of transmit-

TH E M A T U R E central nervous system (CNS) is characterized by a high degree of structural and functional organization. This organization is the result of complex developmental processes, since the cells in the neurotube, which is the source of all CNS neurons, do not appear to be structurally ordered at early embryonic stages (49). Thus, it has been recognized that CNS development is a paradigmatic case of a progressive or constructive series of events involving the growth and maturation of ceils and their organization in functional structures or nuclei, leading to physiological functions (60). Each region of the brain has its own characteristic growth rate and maturaTo whom requests for reprints should be addressed. 413

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ter functions such as the presence of transmitter synthesizing enzymes, of release and uptake mechanisms and of specific receptors, mainly occurs after migration is completed (49). Finally, the time-sequence in which different neuronal cell types and brain regions are developed, is important because neurons a n d / o r regions that are more differentiated, secrete factors (peptides a n d / o r transmitters), which guide and control the development of yet undeveloped groups of neurons and regions (38). Although a good deal of information about the ontogeny of CNS has been obtained by different studies such as physical growth (24,25), biochemical and neurobiological observations of the developing brain (23,38), there still lacks information concerning the chronology of functional maturation of organized structures or nuclei in the CNS. No information is provided by literature about the time course of the regional maturation of brain integrated activities, predicting the spatiotemporal establishment of functionally mature synaptic connections and neuronal circuits during brain development. According to Peters and Jones (62), normal function depends directly on degree of dendritic branchings of neurons and synaptic connectivity. The density of these structural organizations is anatomically difficult to estimate in vitro. The purpose of the present studies was to assess in the rat the overall activity of those organized structures during brain ontogeny. In reaching that purpose, and contrary to in vitro studies (38), we will make more detailed studies, through the ontogeny of psychomotor and sensory functions, of developing functional nuclei and synapses, knowing that the finality of synaptic network is to generate behavior. In order to assess the ontogenetic development of the vast sensory and motor behaviors generated by CNS activity, we will specifically study: the novelty-induced functions (exploratory behavior, habituation, defecation); the motor functions (reflex, automatic and voluntary motor functions). The central problem is to determine when these functions derived from CNS activity come to maturity. Do analyses of structurefunction relationships allow us to reveal the spatio-temporal functional maturation of organized structures and nuclei underlying these functions in the CNS? Consequently, what conclusive hypotheses might be formulated on the time-sequence of regional maturation of brain integrated activities? The following research was performed to address these questions. MATERIALS AND M E T H O D S

Animals Wistar rat pups from rats mated in our colony (from EvicCeba, France) were used. Approximately 1 week prior to parturition, the dams were housed individually in plastic cages (27 x 37 × 18 cm) with the floor covered with wooddust. The dams were checked daily in the morning for pups. The colony was bred in an aerated noiseless vivarium room subjected to diurnal daylight (12 ½ h)/night (11 ½ h) cycles. The colony room was routinely controlled for temperature (25 ___ 1 °C) and humidity (70-80°70). Neonates were pooled within 24 h following birth and litter sizes were adjusted, so that eight to ten pups were randomly assigned per dam. The date of parturition was designated as Postnatal day 1 (P1). After birth, offspring were left undisturbed until 10 days of age. Testing sessions were performed at 10, 15, 20, 25, 30, and 45 days of age. The same pups were assessed from 10th to 45th postnatal day. Mothers remained with the pups at all times, except during testing sessions. At

weaning (21 days), rat pups were housed in cages by sex groups of 3. All animals had access ad lib to CRO lab feed (Centre de Recherche Oc6anographique de C6te d'lvoire) and to tap water. APPARATUS

A battery of apparatuses was used to measure the behavioral parameters of development.

Hole-Board Apparatus The apparatus used was the automatized version of the hole-board first introduced by Boissier and Simon (9). It was a Plexiglas board with a 36 x 36 cm floor and 5.2 cm thick. The board was bored with 16 equidistant holes (4 × 4 holes), each 2,6 cm in diameter. Electric photocells directly placed in the inner side of each hole provided automated measurement of the number of head dip responses by a microcomputer.

String Testing Apparatus The testing apparatus consisted of a piece of iron wire, 0,7 mm in diameter and 37 cm long, tied tightly between two vertical bars and suspended 35 cm over the ground.

Rotarod Apparatus The apparatus is composed of a thick rod (3 cm in diameter) divided into five compartments by circular disks. The rod was turned at a speed of 23 r.p.m, and the rats were forced to walk on the turning rod in an opposite direction. An animal which could not perfectly coordinate its movements failed to walk at the rotation speed of the rod: it was swept along by the rod's movement and fell down. BEHAVIORAL PARAMETERS ASSESSMENT All behavioral testing was conducted between 09:00 and l l : 0 0 a.m.

Hole-Board Test The hole-board apparatus allowed to measure exploratory activity, habituation, emotional defecation and locomotor activity. Exploratory behavior. The hole-board test provides a relatively reliable measure of stimulus-directed exploratory behavior (29). To begin the experiment, each rat was placed singly in the center of the board, facing away from the observer and its behavior recorded for 5 min. Each time the animal dipped its head at least 1,5 cm into the hole, a count was recorded. Only a 5 rain trial was carried out at every age. Habituation. The novelty of the experimental context of the hole-board induces in the rat a response of exploration (8). The decrement of this exploratory response following a prolonged exposure of animal to the same situation was measured. Thus, the number of head dip responses was counted at the 1st, 2nd, 3rd, 4th, and 5th min of exposure to new surroundings. Then, the mean number of head dip responses for each consecutive rain within a 5 min trial of exposure was computed. Only one trial of 5 min was effected at every age. Emotional reaction. The new situation evoked by the experimental context of the hole-board generates anxiety in the animal (8,10). The number of emitted defecations was counted during a 5 min trial of exposure at every age. After each trial, the floor of the apparatus was wiped with

PSYCHOMOTOR FUNCTIONS dilute acetic acid and dried to remove traces of the previous path. Locomotor activity. The space of the hole-board was crossed by two perpendicular luminous rays allowing to cipher the displacement of the animal each time their trajectories were interrupted. The number of crossed rays was recorded during a 5 rain trial by an automatic apparatus. The rays interruptions caused by tail's movements were manually scored and deducted from the total recorded automatically. Only one 5 min trial was performed at every age. The apparatus provides a separate measure of locomotor activity and exploratory behavior (29).

String Test The testing apparatus was used to measure hind paws lifting reflex, wire grasping time, motor initiative, crawling execution latency along the wire and leap execution latency on the ground. Hindpaws lifting reflex. The animal was left gripped by its fore paws at the middle of the wire. The time spent by the animal to retrieve its balance by bringing its hind paws upon the wire, was measured. Wire grasping time. The rat was held by the tail and suspended over the wire near its midpoint. When the rat grasped the wire with its forepaws, the investigator released the tail and began timing. Observation and timing continued until the rat fell off the string. The relatively shorter grasping times were followed by a 2nd, and even a 3rd trial for verification. The grasping times concerned only the periods during which the animal was gripped by the sole forepaws. As soon as it laid down its hind paws on the wire, they were removed by pulling on its tail slightly without pushing over it. Motor initiative. At some time of their development, the rats did not remain gripped at the wire: they would either move sideways towards one of the two poles, or leap onto the ground. We named this behavior: "motor initiative." This test was considered as positive when the animals succeeded in deserting the wire in less than 45s (52). The percentage of animals passing the test was determined. Crawling and leap execution latencies. The rat was compelled to get a grip on the middle of the wire. The time spent to reach one of the two vertical bars by crawling execution, or to leap onto the ground was timed.

Rotarod Test The rotarod performance was used to assess motor coordination. Motor coordination. Each trial consisted of placement of rats on the stationary rod, which, after 15 s, was set in motion. Before the start of each experimental test, the animals were readily trained by conducting three or four trials. Then, the rats were placed on the turning rod in groups of five. The percentage of animals remaining on the rod during a 5 min trial was recorded.

Data Analysis Every behavioral variable was first analysed using one-way analysis of variance (ANOVA). If overall analysis of variance revealed significant differences among population means, differences between individual pairs of means were determined by SheffCs test for multiple comparisons. Motor coordination and motor initiative were assessed by the chi-square test.

415 Pearson correlations were computed between exploratory activity and locomotor activity, between wire grasping time and weight. All results and graphics are presented as means + SEM. RESULTS

Ontogeny of Novelty-Induced Functions Exploratory activity. Our results showed that the development of the exploratory activity in the rat was represented by an inverted U-shaped curve as a function of age (Fig. 1) An overall analysis of variance revealed significant difference among the means of age groups (F 5,115 = 14,675, P = 0.0001). Sheff6's post hoc test ( P's = 0.01) indicated that exploratory activity increased significantly from P10 (10th postnatal day) to P20 (F 1,31 = 7.149) and reached a plateau from P20 to P30 when highest activities were exhibited. At this stage, there was no significant difference between the levels of exploratory activity of 20- and 25-day-old rats ( F 1,31 = 0.046), or 20- and 30-day-old rats (F 1,31 = 0.015). Afterwards, exploratory activity decreased significantly from P25 to P45 (F 1,31 = 3.647). However, the great decrease in activity in 45-day-old rats was partly an artefact, because the perception of the environment would be altered by the experimental context-induced fear. Since 10- and 45-day-old rats did not differ from one another in their activity (F 1,31 = 0.957), at these two extreme ages, activities were somewhat biased. Habituation. In the present experimental context, habituation was not manifest in 10- and 15-day-old rats (Fig. 2). However, habituation was exhibited in 20-day-old rats when large activity decrements were apparent after an exposure time, as short as 4 rain, to the hole-board. The ANOVA on the levels of exploratory activity showed that the means differed significantly at the first minute (F 4,124 = 16.74), second minute (F 4,124 = 12.05), and third minute (F 4,124 = 5.27) of exposure to surroundings (P < 0.005 in the three cases). Habituation has the same rate in 25- and 30-day-old rats. Emotional defecation. An ANOVA on emotional defecation indicated that there were age-related significant changes of emotional reactivity (F 5,115 = 21.185, P = 0.0001). 10and 15-day-old rats showed very little reactivity when they were exposed to new surroundings (Fig. 3). SheffCs analysis for multiple comparisons (P's = 0.01), showed that emotional reactivity developed significantly from PI5 to P20 (F 1,23 = 8.695) and reached a plateau at P20. Indeed, there were no further significant changes from P20 to P45 (F 1,23 = 0.0087). ONTOGENY OF MOTOR FUNCTIONS

Development of Reflex Motor Functions Hindpaws lifting reflex. When the rat gripped a wire by its sole fore paws, it had then an unsteady posture and attempted, in a reflex movement, to return towards a more stable balance by lifting its hind paws and placing them on the wire: we called this behavior the "hind paws lifting reflex." The hind paws lifting reflex appeared early in the rat; the average latency for its occurrence was 2.257 _+ 0.226 s in 10day-old rats, decreased to 0.622 _+ 0.077 s in 20-day-old rats and remained at 0.896 +_ 0.049 s in 45-day-old rats (Fig. 4). The average latency of hind paw lifting changed significantly with age (F 5,110 = 22.07, P = 0.0001). Further examination of this data using SheffCs post hoc test revealed that

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average latency significantly decreased from P10 to P20 (F 1,22 = 14.272, P < 0.01), at which stage it seemed to stabilize. From P20 to P45 there were no further significant changes in latency of hind paw lifting (F 1,22 = 0.401, P > 0.05). Wire-grasping time. During the development, the wiregrasping times increased exponentially in the rat, and the curve really took off by the 20th day (Fig. 5). An ANOVA showed the main effect of age on the measure of wire-grasping times (F 3,75 = 88.716, P = 0.0001). The grasping times were short in 10- (14.112 +_ 1.626 s) and 15-day-old rats

(59.519 _+ 9.851 s), and greatly increased in 20- (202.862 +_ 12.685 s) and 25-day-old rats (517.619 _+ 44.551 s). At 20 days of age, the grasping times were 4-fold higher than in the 15th day (F 1,25 = 5.874, P < 0.01). At the 25th postnatal day, they were 8-fold more important than in the 15th day (F 1,25 -- 59.991, P < 0.01). The exponential evolution of the wire-grasping times is likely to relate to the increase of the animal's weight. However, in our studies, the grasping time correlated negatively with the weight in 15-(r = -0,2199), 20- (r = -0,1926), and 25-day-old rats (r = -0,4529); and both characters were in-

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dependent (p > 0.2 in the 3 cases). In contrast, in 10-day-old rats, the wire-grasping time significantly and positively correlated with the weight (r = 0,6975, p < 0.001).

Development of A utomatic Motor Functions Locomotor activity. The pattern of changes in developing locomotor activity (Fig. 6), exhibited age group differences (F 5,115 = 13.539, P = 0.0001). Scheff~'s post hoc test indicated that this activity developed rapidly from PI0 to P15 (F

1,31 = 4.913, P < 0.01), followed by no significant changes between P15 and P45 (F1,31 = 1.661, P > 0.05). Interesting was the fact that the locomotor activity showed a clean non significant depression, from P15 to P20 (F 1,31 = 0.789, P > 0.05) and to P25 (F 1,31 = 0.694, P > 0.05). This depression was inversely proportional to the increase of exploratory activity (Fig. 7): from P15 to P20, while locomotor activity decreased by 33,3070, exploratory activity increased by 48,1207o. This opposite relation was highly significant between exploratory activity and locomotor activity at the 20th

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