Morphological changes of the pyramidal cell nucleolus and nucleus in hamster frontal cortex during develoment and aging

Mechanisms of Ageing and Development, 15 ( 1981) 385-397

385

© Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands

PYRAMIDAL CELL MORPHOLOGICAL CHANGES OF THE N U C L E O L U S A N D N U C L E U S IN H A M S T E R F R O N T A L C O R T E X DURING DEVELOMENT AND AGING

MB. TANK BUSCHMANN and ARTHUR LaVELLE Department of Anatomy, University of Illinois at the Medical Center, Chicago, Illinois 60612 [U.S.A.}

(Received June 18, 1980; in revised form November 5, 1980)

SUMMARY The development and aging of the nudeolus and nucleus in layer V pyramidal cells in the hamster cerebrum were studied by light and electron microscopy. The nucleoli appeared in the newborn as occasional fibriUar masses adjacent to peripherally placed bodies of chromatin. By maturity, a single, generally central, nucleolus proper with nudeolus-associated chromatin was present. Nucleolar microbodies were observed at 10, 15, 20 and 480 days, but not in the newborn, 5- or 90-day animal. An intranucleolar body was not observed at the electron-microscopy level in these pyramidal cell nucleoli at any age in this series, in contrast to the situation in large motor neurons of the facial nucleus. The nucleus progressed from an irregular shape at birth to an oval shape at maturity. At I0 days, incipient invaginations of the nuclear membrane appeared; these subsequently increased in depth and frequency in the adult. The above changes, particularly in the nudeoli, are correlated in time with changes involving the endoplasmic reticulum. The correlations may indicate different periods of metabolic activity in the hamster pyramidal neurons. Four such periods can be differentiated on the basis of cytomorphic changes which may be correlated to reported development of function. The sequence of these changes, peculiar to the developing and aging hamster pyramidal neuron, differs from that seen in large spinal and cranial motor neurons. It appears that some features of nuclear immaturity, which are lost in larger neuronal types, are retained in the adult pyramidal neuron.

INTRODUCTION Changes in pyramidal neurons are of major significance in the postnatal development of cerebral cortex [1]. In particular it would appear also that developmental and aging changes in layer V pyramidal neurons are significantly involved in the major

386 physiological and chemical events in cortical development and aging. This seems reasonable, considering that the layer V pyramidal cell is in apical communication with all overlying layers and is the most prominent neocortical neuron of projection [2]. In the case of the hamster neocortex, correlations have been reported between stages in the cytological development of these neurons and other events such as the EEG activity [ 3 - 5 ] , the occurrence of endoplasmic reticuhim (ER) specializations [ 6 - 8 ] , and certain aspectsof overt behavioral development [9]. In the present paper, ultrastructural changes in the nucleoli and nuclei of large pyramidal neurons of layer V of developing and aging hamster neocortex are followed. Our particular emphasis is on the nucleolus, which is the most evident nuclear organdie concerned with protein synthesis in the cell. It has been postulated that the attainment of maturity in the nucleus precedes that in the cytoplasm of the cortical neuron [10]. The present observations are suggestive, also, that the pyramidal cell nucleolus and nucleus undergo an extended developmental sequence that results in a retention during maturity of certain features that are lost during maturation in certain other normal neuronal types. Overall, this sequence appears to reflect a succession of functional levels that are correlated with specifically related events in the neuronal perikaryon.

MATERIALS AND METHODS Prior to fixation, newborn, 5-, 10-, 15-, 20-, 90-, and 480-day postnatal hamsters anesthetized with Nembutal (50 mg/ml). Three to four animals per age for a total of 22 animals were used. Fixation was by vascular perfusion according to the method of Peters [11]. The primary dilute fixative contained 1% para. formaldehyde and 1.25% glutaraldehyde in 0.08 M sodium cacodylate buffer (pH 7.3) with 0.02% calcium chloride. This was followed immediately with a concentrated fixative of 4% paraformaldehyde and 5% glutaraldehyde in the same buffer, Both solutions were heated to 40 °C prior to use. Following the perfusion, the animals were left untouched for two hours, after which time the brains were removed from their skulls and were placed into the concentrated fixative overnight at room temperature. Subsequently, a thin transverse slice of tissue was removed from the frontal cortex [12] of each brain. Each slice was then oriented, under a dissection microscope, over a slide-mounted transverse section of hamster cortex of corresponding age stained by a combined Golgi and buffered thionin method [13]. The portion of layer V in the slice was thus isolated by direct comparison with that observed in the stained sections. The isolated, trimmed tissue segments were placed into the concentrated fixative for an additional two hours. The tissue was then washed with a 0.2 M sodium cacodylate buffer (pH 7.3), fixed in 2% OsOa for one hour, washed with the cacodylate buffer, dehydrated in acetone, infiltrated and embedded in Epon-Araldite, and polymerized at 100 °C for one hour. Thick sections were cut for examination by light microscopy at 1 vm with a Porter-Blum MT.2 ultramicrotome. Since pyramidal cells of layer V were not yet recognizable in the newborn, cortical neuroblasts at that age were observed without any

(Mesocricetus auratus) were

387 attempt to identify neuronal type. The large pyramidal neurons in all other ages were identified at the light-microscopic level by their distinctive size and pyramidal shape. The blocks were then further trimmed to include the groups of large pyramidal cells. Thin sections having grey to silver interference colors were then cut for electron microscopy, and stained with saturated, alcoholic, uranyl acetate followed by Millonig's lead tartrate stain [14]. At the ultrastructural level pyramidal cells were distinguished by their lack of asymmetrical axosomatic synapses from nonpyramidal neurons [15-18]. Both examination and photography of the sections were done on a Philips EM-300 electron microscope which was calibrated with a diffraction grating. Lamellar body length and cisternal number at 10, 20 and 480 days were recorded by the method described elsewhere [ 7 ] . Percentage of cells containing lamellar bodies and microbodies were based on cell profiles as observed in electron micrographs. Lamellar body (LB) length, number of cisterns and number of microbodies (MB) per nucleolus were analyzed with the Student-Newman-Keuls test and written as mean values + S.E. The significance of change in frequencies with age was evaluated with the Chi-square test. The number of microbodies per nudeolus was only determined in nuclei containing microbodies. The nuclear and nucleolar apparatus diameters were measured on camera lucida drawings made from thick plastic sections. Two perpendicular diameters were averaged for each nucleus and nucleolus profile and for each animal the average caliper diameter was determined. From this caliper diameter the real diameter was calculated by 4_ = - - d where /9 = real diameter and d = caliper diameter average. The statistical n, 7r

therefore, was the number of animals per age.

OBSERVATIONS

Nucleolar development In the pyramidal cell nucleus of the newborn, two to three large condensed masses of chromatin, consisting of an homogeneous granular material, were mainly peripheral (Table I) and caused bulgings of the nuclear envelope out into the cytoplasm (Fig. 1).

CHANGES IN NUCLEAR CONTOUR WiTH AGE

N~wDorn

20 Dayl~

5 DCF=

POOaFs

90 Dayl=

~50oyl

480 Dayl

Fig. 1. Camera lucida drawings of nuclei typical for each age. Note the irregular nuclear contour with bulging chromatin bodies in the newborn and the generally oval nuclear shape with invaginations at 20, 90, and 480 days. (X 318).

8.04 f 0.24

1 14 + 0.04

2.00 f 0.07

Nuclear diameter (Mm)

NA position*

No. of NA per nucleus

1.22 * 0.50 1.80 f 0.87

2.05 + 0.75 9.25 * 1.27

S days

1.60 +_0.05

1.53 + 0.04

2.61 f 0.07 14.16 + 0.05

10 days

1.40 r 0.06

1.57 + 0.05

2.63 _+0.09 15.10 It 0.25

15 days

1.40 + 0.05

1.77 + 0.06

2.94 + 0.20 14.94 f 1.52

20 days

1.10 + 0.05

3.11 f 0.11 12.45 f 0.74 2.12 + 0.06

90 days

1.20 + 0.04

2.63 f 0.11 11.34 t 0.34 1.51 2 0.06

480 days

From birth to maturity, the nucleolar apparatus (NA) increases in size and moves from a peripheral to a central position. The total number of NA decreases to the, usually, single one found in the adult nucleus. Measurements expressed as mean values f S.E. from 3 animals per age. Counts averaged 50 nuclear and 48 NA profiles per animal per age. *Arbitrary values were assigned to NA position such that 1 = peripheral, 2 = off center and 3 = central.

1.81 f 0.01

NA diameter (Mm)

Newborn

DEVELOPMENT OF NUCLEOLAR APPARATUS

TABLE I

389

Fig. 2. Nucleoli in pyramidal cells. (a) At 5 days of age, consisting mainly of nucleolonema (arrow) with light areas (arrowhead) and few scattered granules. (X 21 700). (b) At 15 days of age, two nudeoli with mierobodies. Note also the two invaginations of the nuclear envelope (arrows). (× 7 400). (c) High magnification of larger nucleolus contains the greatest frequency ofmicrobodies (MB) observed. (× 21 700).

390 Electron-dense fibrillar material, occasionally containing a few granules, appeared in close apposition to some of the chromatin bodies. The fibfillar material most likely represented the early appearance of nucleolar substance. By 5 days, the two or three peripherally located chromatin bodies no longer caused a bulging out of the nuclear envelope (Fig. 1). Nucleolar substance, largely consisting of a dense, felt-like fibrillar material (nucleolonema), was now more regularly associated with some of the chromatin bodies. Finely fibrillar material was also organized into light areas and intermingled in the nucleolonema (Fig. 2a). Similar light areas surrounded by the nucleolonema of nucleoli of mouse neurons were presumed to be the nucleolar organizer [19]. The combination of the nucleolus-associated chromatin and the nucleolus (nucleolonema fibriUar strands as well as light areas and granular constituents) is hereafter termed the nucleolar apparatus (NA). From this time on to 90 days postnatally, there was a very gradual decrease in the number of nucleolar apparatuses (Table I) per nuclear profile. At 10 days, the nucleolar apparatuses were positioned usually peripherally within the nuclei. The scattered granules (14-17 nm in diameter) associated with the nucleolonema appeared more discretely defined than at the preceding two ages. Enmeshed within the nucleolonemal strands of some nucleoli (2 I% Of the profiles viewed, Table II) peculiar spherical structures (Fig. 2b, c) were seen which resembled the "microspherules" described by Busch and Smetana [20]. These structures, here termed microbodies, were scattered and few in number as compared to those present in the 15-day nudeolus. Because Busch and Smetana [20] identify two morphological forms which they term "microspherules" (some are like those described here and others are plaque-like, nonspherical and lack halos), we have used the term microbodies to avoid confusion. The nucleolar apparatuses of 15-day neurons were mainly peripheral in position. The quantity of nucleolar substance proper was predominant in proportion to that of the nucleolus.associated chromatin. Within the nucleolar substance, the nucleolonema consisted of a tangle of strands of fibrillar material. Light areas as well as granules were interspersed among the strands. The rounded light areas of fibriUar substance intermingled with the nucleolonemal strands were comparable to those seen at 5 and 10 days. The microbodies observed first at 10 days were again seen within the nucleolonemal strands of some nucleoli (Fig. 2b, c). These microbodies consisted of round dense material (65-75 nm in diameter) surrounded by an electron-transparent halo. The dense material was similar in character to the fibrillar component of the nueleolonema. The frequency of occurrence of the microbodies appeared to peak at 15 days (44% of profdes, Table II and Fig. 3). By 20 days, nucleolar apparatuses were more frequently located in or near the center of the nuclear profiles. As seen previously, the nucleolonema with intermingled light areas was again observed at 20 days and at all subsequent ages. At this age, only three nucleolar profiles in the 61 pyramidal cell nuclear profiles examined were observed to contain microbodies (Table II, Fig. 3). The adult nucleolar apparatuses at 90 and 480 days were mostly central within the neuronal nuclei (Table I), in contrast to those at the younger ages. The nucleolus-

0 (0%) 0+-o 0+-o

134 0 (0%) o+o 0+-o

127

0 (0%) o+o

143

0 0%) o+o

228

16 (27%) 0.80 f 0.11 2.40 + 0.11

60

14 (21%) 3.90 f 0.11

68

10 days

47 (68%) 1.36 + 0.09 3.10 r 0.10

69

28 (44%) 4.20 ?r 0.77

64

15 days

4 (7%) 0.30 It 0.04 2.00 + 0

54

14 (21%) 0.79 + 0.06 2.10 + 0.04

66

0 (0%) o*o

112

61 3 (5%) 4.00 f 1.53

90 days

20 days

2.10 _+0.03

33 (49%) 0.50 * 0.03

68

6 (12%) 2.50 + 0.76

52

480 days

This table illustrates the nucleolar microbody (MB) and cytoplasmic lame&r body (LB) peaks at 15 days postnatally and their significant increase again at 480 days. At 15 and 480 days the frequency of cells containing MB and LB was significantly different from that of the other ages 0, < 0.001). The IS-day LB length was significantly different from that of the other ages 0, < 0.05).

Average length per LB brn) Average No. cisterns

Profiles with LB

Total No. cell profiles

Promes with MB No. MB per nucleolus

Total No. nucleolar profiles

5 days

Newborn

MICROBODIES AND LAMELLAR BODIES

TABLE II

392 70a=

[

]

%

CELLS WITH LAMELLARBODIES ~'~% NUCLEOLIWiTH MICROBODIES [ ] % LENGTHOF INVAGINATEDNUCLEAR ENVELOPE

50==

% :3Din

IOtu

I-V 5 IO 15 L:"390 48O

5 io 15 zoso4eo

5 Io is zo9o48o

AGE IN DAYS

Fig. 3. Morphologicalchanges as a function of age. Note the significant increase in the frequency of lamellar bodies and microbodies at 15 days and 480 days, whereas invaginations of the nuclear envelope progressivelyincrease from 10 days to 480 days. Chi-squared test, p < 0.001.

associated chromatin was positioned adjacent to the nucleolus. Granules were generally interspersed among the nudeolonemal strands, but no nucleolar microbodies were observed at 90 days (Table II, Fig. 2). Intranudeolar bodies (very large condensed aggregates of ribonucleoprotein particles) [21] were not observed in the pyramidal cell nudeoli examined at any age in this series. Correlated with the peak incidence of microbodies at 15 days and their reappearance at 480 days was the high inddence of lamellar bodies in the cytoplasm at these two ages (Table II, Fig. 3) and also as described elsewhere [7, 8].

Nuclear envelope changes Newborn neuroblast nuclei were very irregular in shape (Fig. I). By 5 days postnatal age, nuclei were usually oval to round in shape and had negligible indentations of their envelopes. At 10 days postnatally, the nuclei were usually round in profile and indentations were rare. At this age the average nuclear diameter was somewhat greater than adult dimensions (Table I). In the 15-day neuron, the nuclear contour in contrast to the previously smooth, nuclear profiles, began to show more pronounced irregularities that apparently presaged the distinct invaginations which appeared later (Figs. 1, 2b and 3). By 20 days postnatal age, nuclear profiles, which were generally oval, showed occasional indentations or distinct invaginations. The neuronal nuclei of the 90-day and 480-day adult were also oval in shape and showed invaginations that were deeper and more frequent than those a t the previous ages (Figs. 1 and 3). A more complete statistically oriented description of the nuclear envelope changes during later aging will be presented elsewhere [22].

393 DISCUSSION Major cytomorphic events in the postnatal development of the large pyramidal cell nuclei of layer V involved the elaboration of the nucleolar apparatus, the occurrence of nucleolar microbodies, changes in number and depth of nuclear envelope invaginations and changes in nuclear shape [8, 23]. The sequence of these changes could be subdivided into at least four fairly well defined periods, the first three of which have been described earlier with reference to cytoplasmic changes [5, 7]. During the first period, from birth through 10 days postnatally, the nuclear profiles containing bulging chromatin bodies peculiar to the newborn became smooth in contour, and the number of chromatin bodies decreased. Fibrillar and granular constituents of the nucleolus markedly increased while closely associated with chromatin bodies. The fibrillar elements were at first more predominant than the granular. There is suggestive cytological evidence that nucleolar fibrillar elements are precursory to the granules [24-27]. Recent biochemical studies of isolated nucleolar fibrillar and granular components also indicate that the fibrillar material is precursory to the granules [28]. The progressive, parallel increase in nucleolar constituents and in nuclear and cytoplasmic diameter of hamster pyramidal cells during the later part of this period is similar to that described, at the lightmicroscopic level, for various developing neurons in the central nervous system of the guinea pig and hamster [I 3, 29-31]. Invaginations of the nuclear envelope were rare or absent during this first period. The second period, from 11 to 19 postnatal days, is typified by the 15-day pyramidal neuron, which is of particular interest in this series. The average nucleolar apparatus at this time was becoming larger and was moving more centrally in the nucleus. Nucleolar microbodies were more frequent than at any time. Bodies morphologically similar to these and termed microspherules have been described in both highly metabolic ceils (for example, neoplastic cells) and in drug-inhibited nucleoli [20]. The first definitive invaginations bf the nuclear envelope have appeared. These unique events at 15 days of age may be related to the fact that this is a pivotal period of transition from immaturity to maturity. In the cytoplasm the stacking of the rough endoplasmic reticulum (RER) cisterns and the size and frequency of lamellar bodies, specializations of the RER, are most pronounced at this age [6-8]. The basal dendritic spread and spine development and nuclear and somal volumes have attained adult proportions [5, 13]. Myelination has already begun in the forebrain of the hamster [32] and. the adult pattern of EEG activity is present [3, 4, 9]. Behaviorally, the hamsters' eyes are opening, weaning is in progress, and there is the first evidence of directive movement [9]. It would appear that the 15day neuron is in a state of changing physiochemical demands. Correlated to meeting these demands is the presence of nudeolar microbodies, lamellar bodies and definitive nuclear envelope invaginations. These features occur in organdies which are not yet adultdike in form and/or number. Furthermore, oligodendrocyte satellite relationships have not yet been established [33]. The third period involving the establishment of maturity, includes the ages 20 days postnatal and the 90-day adult. By 90 days the nucleolar apparatus diameter has reached

394 a maximum and microbodies have disappeared. The depth of nuclear envelope invaginations continued to increase. The complexity and degree of stacking of the RER cisterns into Nissl bodies actually decreased. In respect to these features as well as to further nucleolar and nuclear development in pyramidal cells, the sequence of organdie development cannot be generalized with that already observed for large radicular motor or sensory neurons [34, 35]. For example, the pyramidal cell nucleolar apparatus did not attain the degree of cytological development seen in large, somatic motor neurons of the peripheral nervous system as already noted for the guinea pig [29, 30]. The sequence of organelle development undergone by the pyramidal ceils strongly suggests that qualitative, stage-specific changes in neuronal metabolism are being reflected. The 480-day animals apparently indicate the existence of a fourth period of pyramidal cell change. It should be noted that, according to the National Institute on Aging (Dr. D. Gibson, personal communication), the definition of "aged" is any age older than the average life span of an animal. Since the average life span for the hamster is 1.6 years or 584 days [36], our oldest hamsters can be considered as approaching old age. At this time there is again a significant increase in lamellar bodies, a reappearance of nucleolar microbodies and the continued increase in the degree of nuclear envelope invaginations. In the cortex of other aging animals it has been reported that there is a decrease in the number of neurons [37, 38], of synapses [39], of dendritic branches [40], of ribosomes [41] and in the amount of extracellular space [42]. Assuming this could also be true for aging hamster cortex, then the pyramidal neuron is again associated with a period of changing physiological demands which may be peculiar to the aging process, in contrast to the maturing 15-day neuron. The intranucleolar body described for motor neurons of spinal and cranial nerves in the hamster was not seen in the pyramidal cells during any of the four periods of our present ultrastructural series. With light microscopy, however, it was seen occasionally in pyramidal cells of the hamster [43]. In large motor neurons, the body appears relatively late in development, after the formation of Nissl bodies, and disappears during chromatolysis. It has been suggested that the presence of this intranucleolar body, a duster of ribonucleoprotein granules, is indicative of a metabolic "idling" mechanism wherein a reverse of ribonucleoprotein is being maintained [21,44]. Therefore, the normal absence of this intranucleolar body in pyramidal cells during maturity might relate to a continuation of certain features of metabolic activity otherwise typical of rapidly growing neurons. The subsequent decrease in RER cistern stacking and the lack of nucleolar microbodies in the third period also suggest that this may be the prevalent metabolic condition. Considering these points, we would speculate that the pyramidal neurons do not undergo a complete phasing out of all developmental characteristics of metabolima during maturation. Because nuclear invaginations are frequently observed in rapidly growing and injured neurons, they may function in heightened nuclear-cytoplasmic exchange in neurons [45, 46] as well as in other types of cells [47, 48]. These invaginations were not seen in the hamster pyramidal cells when their perikaryal growth was most rapid, in contrast to their presence in large motor neurons during their rapid growth phase. Significant qualitative differences are therefore probably involved.

395 In general, the peculiar sequence of morphological changes observed in the developing nuclei of pyramidal cells of the hamster is strongly indicative that the high level of metabolic activity initiated during the middle part of the first period gradually undergoes a specialization of output, but nevertheless persists during maturity and aging. It appears that this change in activity is also correlated with organelle changes in the cytoplasm in which stacking of RER cisterns and lamellar body size and frequency decreased during the third period [6-8]. It has been suggested that in whole brain a microsomal system concerned with the synthesis of structural proteins may be phased out during maturation [49] and that stable messenger RNA-ribosomal complexes in developing cerebral cortex are replaced by unstable messenger RNA complexes concerned with the production of labile proteins [50]. In hamster pyramidal cells, as reported here, the decreased stacking of the RER cisterns, the reduced lamellar body frequency and the disappearance of nucleolar microbodies observed during the third period appear concordant with these biochemical studies. The changes in the nuclear envelope in the pyramidal neurons, on the other hand, may not be precisely accounted for by the same biochemical changes involving the total cell population of whole brain or cortex. The layer V pyramids are the most pervasively involved neurons in cortical function. The cytomorphic phases of their nudeolar and nuclear development probably reflect stage-specific levels of neuronal activity which appear over a prolonged period of maturation and reflect their participation in cortical functions. At the present time, however, it is not understood how specific changes in organeUes contribute differentially or reflect the total biochemical state peculiar to each successive period. In the developing hamster facial nucleus, certain nuclear stages indicate sensitive phases in terms of reaction to injury [51]. Similarly, the nuclear states described for the layer V pyramidal neurons may indicate sensitive developmental and aging periods for these cells. Experimental investigation is needed to clarify this line of speculation.

ACKNOWLEDGMENTS This research was supported by Public Health Service Grants NU 5020, BRSG 5776-4 and GRSG 503. We wish to thank Karen DeMello for typing the manuscript, Janet N. Meyer for the illustrations, and Dianne Chong for technical help.

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