ISSN 00220930, Journal of Evolutionary Biochemistry and Physiology, 2008, Vol. 44, No. 1, pp. 109—115. © Pleiades Publishing, Ltd., 2008. Original Russian Text © T. L. Oleinik, R. A. Grigoryan, 2008, published in Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, 2008, Vol. 44, No. 1, pp. 94—99.

MORPHOLOGICAL BASICS FOR EVOLUTION OF FUNCTIONS

Postnatal Development of the Shape of Cerebellar Purkinje Cells in Guinea Pig Ontogenesis T. L. Oleinik and R. A. Grigoryan Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia Email: [email protected] Received November 28, 2006

Abstract—In sagittal cerebellum sections, morphometrical study of cerebellum of matureborn an imals—guinea pigs—was performed using Nissl’s procedure. A change of shape and volume of Purkin je cells and their nuclei in the course of the guinea pig postnatal ontogenesis was studied. It has been shown that both the growth process itself and the rate of formation of the definite form of Purkinje cells and of their nuclei in the course of ontogenesis proceeds nonuniformly. The most intensive growth of vertical and horizontal diameters of Purkinje cells and of their nuclei is observed during the 1st and 4th weeks of postnatal life. Especially rapid is an increase of horizontal diameters of Purkinje cells and of their nuclei, which impairs the ovoidbearlike shape to the cerebellar Purkinje cells of adult guinea pigs. DOI: 10.1134/S0022093008010131 Key words: cerebellum, Purkinje cells, guinea pig, ontogenesis.

INTRODUCTION One of fundamental problems of evolutional neurophysiology is study of regularities of the brain maturation at the process of ontogenesis. Cerebel lum as a brain part represents a peculiar structure in the central nervous system. Its peculiarity con sists in that apart from the control and coordina tion of movements, it participates in sensory–mo tor integration, perception of auditory, visual, and visceral information, in leaning of motor skills. Cerebellum has been shown experimentally to be involved in cognitive function [1]. At the same time, strange as it is, until now the most part of these cerebellar functions have been studied insuf ficiently. We started studying size, shape, and volume of Purkinje cells (PC) not by chance, but because

these cells are the only output element of the cere bellar cortex; we considered it of interest to study development of their definitive form in represen tatives of various animal species that have differ ent terms of maturation of the cerebellum func tion. Besides, a study of size and shape of the PC soma also is of a great importance due to that to pology of synaptic contacts on PC dendrites has a great significance for efficiency of the synaptic ac tion; especially important is contact of PC den drites with the basket cell that appears in phylo genesis only in birds and mammals [2]. It is to be noted that in birds, practically almost the entire Purkinje cell soma obtains synaptic contacts from the basket cells, which seems to be important for the rapid and precise movements used by birds first of all during the fly and soft landing. The Purkinje cell is the largest cerebellum cell (and one of the

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largest in CNS) with the abundant branched den drite tree; it is therefore that it contributes the most to the whole cerebellum volume. For example, it has been shown, that the Purkinje cell number as well as their volume, size, and the way of innerva tion plays an essential role for elaboration of the nictating conditional reflexes [3]. A high correla tion exists between the Purkinje cell volume and the rate of elaboration of the human and animal conditional reflexes [4], i.e., its volume is function ally important for such form of associative learn ing. At our laboratory, we have already studied the Purkinje cell shape and sizes in immatureborn animals—rats and cats [5, 6]. Therefore it was of interest to compare them with the data obtained in the matureborn guinea pigs. MATERIALS AND METHODS In our work, 5 age groups of the studied guinea pigs were used: newborn, oneweek, twoweek, threeweek and onemonth. A morphometrical study was performed in each group of 8 animals. Thus, the studies were performed on 40 guinea pigs. It has been shown preliminarily that the gender differences in the cerebellum structure in this ani mal species are absent, as there was no statistically significant difference between the Purkinje cells in the guinea pigs of different gender. Cerebellum was removed under urethane narcosis and fixed in a formalin and alcohol mixture, then paraffin sec tions were prepared and stained using Nissl’s pro cedure. For measurements and further statistical treatment, sections from 3 cerebellum parts were taken: from the lateral and medial parts of the hemisphere and from the middle part of the ver mis. In the animal, 30 sections were analyzed from each of the above cerebellum parts, 90 cells in each section. The results of measurement were com pared and evaluated statistically by Student’s cri teria. In all cases, differences in sizes of PC and their nuclei in different cerebellum areas in the same animal were statistically nonsignificant. Thus, the result was averaged from 90 sections of one cerebellum in one individual and then was av eraged for all animals of the age group. The Purkin je cell diameter and the cell nuclei were measured using a device on the microscope eyepiece at 600× magnification. The volume of both Purkinje cell

and its nucleus was calculated [7]. Taking into ac count that Purkinje cell in the definitive state has the pearlike shape, both horizontal and vertical diameters were simultaneously measured in the same cell, as their ratio provides a more correct information about the relative change of the cell and nucleus shape for the first month of postnatal development. For convenience of comparison of the previously obtained data about the change of Purkinje cell siz es in animals of different species (guinea pigs, rat pups, kittens), we performed some calculations showing more clearly an increase of the cell sizes in the processes of postnatal ontogenesis [5, 6]. The mean Purkinje cell diameters in newborn animals were taken as 100%; the diameters at other peri ods of the postnatal life were expressed in percents relative to the newborn animals. Unfortunately, it is rather difficult to compare the data about Purkinje cell sizes and nucleus diame ters, obtained by different authors. The peculiarity of the morphological studies is a marked depen dence of the obtained quantitative values on the chosen procedure due to effect on the tissue of dif ferent fixing and dehydrating fluids, of the way of embedding of the material, etc. The present work allows comparing sizes of Purkinje cells and their nuclei at various stages of ontogenesis with the data obtained earlier on rats and cats, without calculat ing the coefficient of the tissue consolidation or swelling during fixation, shrinkage, and elongation as a result of further treatment, as all studies were performed using the common scheme [5, 6]. RESULTS This work was performed on the classic labora tory representative of matureborn animals—guin ea pig. According to the literature data, the com plete cerebellum maturation in immatureborn animals (rat, cat), unlike the matureborn ones, occurs by the twomonths period of the postnatal ontogenesis [8]. Based on our data obtained by the light micros copy method, it can be stated that in newborn guin ea pigs, cerebellar Purkinje cells have contours of the ovoid or droplike (pearlike) shape with well identified large and light nucleus with the clearly seen dark basophile nucleolus. In two out of eight

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Preparations of guinea pig cerebellum (Nissl’s staining) at various stages of ontogenesis. Guinea pig: (a) newborn, (b) the 7day, (c) the 14day, (d) the 30day old animal. PC—Purkinje cell, ML—molecular layer, GL—granular layer. Scale bar 10 μm.

studied individuals, the Purkinje cell bodies were located only inside sulci with some deviation from the horizontal axis up and down, which partly agrees with literature data [9]. The Purkinje cells in other individuals formed a regular monolayer, which distinguishes our data from the data ob tained by other authors reporting the Purkinje cell location in several layers. Approximately the same picture was observed in the earlier studied imma tureborn animals [5, 6], but their Purkinje cells had a more intensive color under the similar stain ing conditions, than in guinea pigs of same age. The Purkinje cell bodies were inserted deeper into the lower layers of the cerebellar cortex and were ar ranged in all individuals in the clear line relatively to each other. The cerebellar molecular layer was less thick as compared with guinea pigs, whereas the external, granular layer, on the contrary, was more expressed and tightly packed.

The Purkinje cell vertical diameter in newborn guinea pigs was 19.2 ±0.16, while the horizontal diameter 14.2 ± 0.15 μm. The cell volume was 2585 ± 626 μm3. Figure 1a represents a photo of the cerebellum sections in newborn guinea pigs. Clearly seen are Purkinje cells with the light nu cleus and nucleolus as well as the cerebellar mo lecular and granular layers. Purkinje cells were ar ranged as one horizontal line, i.e., formed a well identified monolayer. A significant increase of the Purkinje cell vertical diameter (1.12 times) oc curred in the oneweek animals (see Fig. 1b), while the horizontal diameter varied less (1.06 times). On the whole, the Purkinje cell volume increased to 1.33 times one week after the birth (Tables 1 and 2). Parameters of both diameters increased relative ly uniformly for the 2nd and 3rd weeks of the post natal life.

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Table 1. Sizes of cerebellar Purkinje cells and of their nuclei in ontogenesis of matureborn animals

Animals

Age (days)

Guinea pig

Purkinje cell diameter

Purkinje cell nucleus diameter

vertical

horizontal

0–1

19.2 ± 0.16

14.2 ± 0.15

11.0 ± 0.16

9.5 ± 0.18

7–8

21.5 ± 0.18

15.1 ± 0.17

11.8 ± 0.2

10.2 ± 0.19

14–15

22.5 ± 0.18

15.8 ± 0.16

13.2 ± 0.17

11.0 ± 0.14

21–22

23.4 ± 0.18

16.8 ± 0.15

13.6 ± 0.18

11.4 ± 0.17

30–31

25.2 ± 0.20

20.3 ± 0.19

14.0 ± 0.2

11.8 ± 0.14

Table 2. Volume of cerebellar Purkinje cells and of their nuclei in ontogenesis of matureborn animals Animals

Age (days)

PC volume (μm3)

PC nucleus volume (μm3)

Guinea pig

0–1

2585 ± 62.6

671 ± 19.2

7–8

3422 ± 73.4

751 ± 19.9

14–15

4100 ± 81.4

976 ± 21.4

21–22

4420 ± 89.8

1165 ± 20.6

30–31

6487 ± 85.3

1279 ± 19.7

The cerebellar Purkinje cell vertical diameter in guinea pigs by the 21st day of the postnatal devel opment was 23.4 ± 0.18, while the horizontal one 16.8 ± 0.15 μm. Thus, this period, both diameters increased almost equally—1.2 times. By the 30–31st day of the postnatal life, both vertical and horizontal diameters of Purkinje cell in guinea pigs continued to increase. However, whereas the vertical diameter increased 1.3 times as compared with the newborns (an increment was 1.8 μm), the horizontal one—1.4 times (an incre ment was 3.4 μm). Thus, the vertical diameter of Purkinje cell was increased during the first month of life by 6, while the horizontal diameter by 6.1 μm. This increase is well demonstrated in mi crophotograph (see Fig. 1d) from the cerebellum morphological preparation of the onemonth old guinea pig. As already noted in “Materials and Methods,” the Purkinje cell mean diameters in newborn ani mals were taken as 100% and were compared with the diameters at the subsequent studied periods. Based on these calculations, the lowest rise of both

vertical

horizontal

vertical and horizontal diameters of Purkinje cells was observed in the guinea pigs, as compared with the earlier studied animals. The vertical diameter had two increase picks—by the 7th and 30th days of the postnatal life (on the whole by 43%), the horizontal two picks—by the 21st and 30th days (on the whole by 31%). The horizontal diameter in creased the most (by 95%) in rats by the 14th and 30th days of development (by 16 and 42% as com pared with the newborn animals, respectively). The vertical diameter in the rat pups varied less mark edly (by 49%), but had two picks—by the 14th day and by the abovementioned period (the 30th day). The vertical diameter increased in kittens by the 30th day by 59%, which is the highest value for this parameter; it varied the most by the 21st day of the postnatal life. The horizontal diameter increased on the whole by 82% and had the increase pick at the 30th day. DISCUSSION In this study the results were obtained, which it is of interest to be compared with our earlier date on kittens and rat pups, i.e., on immatureborn animals [5, 6]. The obtained morphometrical pa rameters about terms and rates of acquisition of the definitive shape and size of the cerebellar Purkinje cells in mature and immatureborn animals agree on the whole with terms of functional maturation in these animals of the main afferent inputs—sys tems of the mossy and climbing fibers to the cere bellar Purkinje cells as well as of such statokinetic reflexes, as reflex of standing, supporting, and lift ing reactions [10]. When the cerebellar Purkinje cells reach the

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shape and sizes characteristic of adult animals, the functions of the standing, walking, and postural motor reactions approach by their characters those in adult animals, i.e., there is a close interrelation between terms of histogenesis of the cerebellar cor tex, functional maturation of electrophysiological parameters of the Purkinje cell activity and acqui sition of their definitive form as well as tempo of realization of statokinetic reflexes. The cerebellum morphogenesis in kittens and rat pups occurs mainly after birth, always takes a longer time (about two months), and has the slower development, as compared with guinea pigs. In this connection, it is to be noted that the terms and rates of morpho logical maturation of the guinea pig cerebellum in ontogenesis, strange as it is, have remained poorly studied until now; however, there are data that guinea pigs are born with a more mature cerebel lum, both morphologically and functionally, as these animals can stand and walk as early as at the 1st day after their birth [11]. In ontogenesis the process of complete matura tion of the cerebellar Purkinje cell shape in guinea pigs takes about 3–4 weeks, in rats 4 weeks, and in the kittens it is longer and takes about 6–7 weeks of the postnatal life [10]. It is to be noted that our morphological prepa rations of the guinea pig cerebellum not completely agree with literature data. By the opinion of au thor of the work [9], Purkinje cells of adult ani mals are arranged in 2–3 stores and are inserted partly into the granular layer. But our data indi cate that as early as by the 7th day of postnatal de velopment they are already arranged in the well formed monolayer without any deviation from the horizontal line (see Fig. 1b). It is also to note the brighter staining of kitten Purkinje cells at the first week after birth as compared with rat pups and guinea pigs that have the lightest PC body under similar conditions of fixation and staining. This indicates a metabolic PC activity in immature born animals at this ontogenesis period and about a peculiar protein “overproduction” due to forth coming cell growth. One of the most important parameters of the cerebellum morphological maturity is the period of completion of migration of granular cells from the external into the internal layer and of reduc tion of the external granular layer [12]. It has been

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shown in several works that this histogenesis peri od coincides with functional maturation of Purkin je cells, which is characterized by certain electro physiological correlates (an increase of the PC fire frequency), and with period of establishment of the activity parameters [10, 13, 14]. Since afferent sig nals from mossy fibers come to PC through granu lar cells, their neurogenesis is of crucial significance for the Purkinje cell activity. An important pecu liarity of the cerebellum cortex neurogenesis is that the granular cell migration occurs at the initial pe riods of the postnatal development from the exter nal into the internal layer [9, 15, 16]. According to literature data, this migration process in guinea pigs is completed by the 6th day of postnatal life [17] and in rat pups to the 21st day [12], while in cats it is longer and is completed by the 35–60th day of life [6, 16, 18]. Our data also indicate an even fast er process of the disappearance of the external granular layer both in rat pups [5]—to the 14th day and in guinea pigs—to the 7th day (see Fig. 1c). Although study of the granular cell migration was not the goal of this work, still examination of prep arations has detected that the external granular lay er in newborn guinea pigs consists of occasional cells and disappears completely as early by the 3rd day of postnatal development. At our laboratory, by using electrophysiological methods, problems of the Purkinje cell functional maturation in guinea pigs have been well studied and it has been shown that the complete function al cerebellum maturation occurs in these animals by the 3–4th week of postnatal life [13]. In rats this period continued until the 4th, while in cats until the 7th week [10]. Thus, comparative short terms of maturation of the mossy output afferents into PC in guinea pigs are due to the sufficiently fast completion of mi gration of granular layer elements, which began at the embryonic period, while at the postnatal allows performing activation of the synapse “parallel fi ber– Purkinje cell” [16, 18–20]—the basic main element participating in control of the postural motor reflexes [21]. Acquisition by the Purkinje cell of definitive shape is related naturally to its dendrite tree for mation, particularly that the stained cytoplasm at the dendrite base has clearly seen in the obtained morphological sections. It can be judged about the

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level of the Purkinje cell dendrite arborization from its shape, specifically from thickness of the den drite base, which is well stained and clearly seen under microscope due to the cytoplasm flow. This process characterizes a sharp increase of the Purkinje cell horizontal diameter, as it is accom panied by the cytoplasm redistribution within the soma limits. In summarizing all the above and in comparing the rate of the Purkinje cell shape development in guinea pigs, rat pups, and kittens, it can be con cluded that PC in the guinea pigs experience the lowest changes, as their cerebellum initially is more developed morphologically; Wistar rats and cats have less mature PC, their diameter increase fast er and the highest maturation rate is observed at the third and forth weeks of postnatal development. Based on the obtained data, it can be concluded that morphological maturation of the Purkinje cell shape and volume occurs differently in early onto genesis in three studied animal species. First of all, it is to be noted that it is the horizontal diameter of Purkinje cells in all animals, which changes the most, as the cell is initially elongated in the plane perpendicular to the cerebellar folium. Acquisition of the definitive pearlike shape leads to an increase of the transverse diameter of the PC soma. Inter estingly, this diameter in rats and kitten increases 2 times, while in guinea pigs only 1.4 times. The change of the horizontal diameter can be ascribed to formation of the synaptic contacts on the PC body, first of all with basket cells. According to lit erature data, these contacts in rats appear by the 14th day of postnatal development [13], and it is at this period that the first peak of the horizontal di ameter increase is observed [5]. The vertical diam eter increases less intensively, but this increase in dicates elongation of the cell soma due to the den drite tree growth and formation of its contacts with parallel fibers. At the same time, it is to be noted that a rela tively greater, 5fold increase of the PC volume occurs in kittens as compared with guinea pigs and rats, while in representatives of rodents the increase is about 2.5fold by the age of 30 days. It can be suggested that the great volume increase leads to the more intensive intracellular metabolism and promotes performance the greater assortment of movements [13, 14].

ACKNOWLEDGMENTS The work is supported by the Russian Founda tion for Basic Research (project no. 060449428). REFERENCES 1.

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