Age-related changes in the neuropil in the rat inferior olive nucleus: A quantitative electron microscopic study

BrainResearchBulletin, Vol. 8, pp. 381-388, 1982. Printedin the U.S.A.

Age-Related Changes in the Neuropil in the Rat Inferior Olive Nucleus: A Quantitative Electron Microscopic Study KOICHI

IKARI,

MICHIHIKO

HAYASHI

AND RYO OGATA

Department of Neuropsychiatry, Faculty of Medicine, Kyushu University, I-I, Maidashi I-chome, Higashi-ku, Fukuoka 812, Japan Received

I July

198 I

IKARI, K., hf. HAYASHI AND R. OGATA. Age-related changes in the neurapil in the rat inferior olive nucleus: A quantitative electron microscopic study. BRAIN RES. BULL 8(4) 381-388, 1982.-Age-related ultrastructural changes in the neuropil in the rat inferior olive nucleus were examined at 3, 6. 12, 18, 24 and 30 months old. The profiles of axon terminals, dendrites and nstroglial processes from random samplings within the neuropif were traced. Subsequently, the percentages of these pro&d areas in relation to the area of neuropil (relative volume fraction) were examined using the image analyzer system. The relative volume fractions of both axon terminals and dendrites in relation to the neuropil were found to have decreased in the aged rats, while the relative volume fraction of astroglial processes had progressively increased with aging. Inferior olive nucleus

Aging

Quantitative electron microscopic study

THE inferior olive in the human brain, one of the li~p~~ic nuclei, contains lipofuscin granules (aged pigment) in the cell soma beginning in the fetal stage [3]. The intracellular accumulation of lipofuscin granules were reported to be correlated directly with aging in this nucleus [21]. Mann and Yates [20) proposed that cell death may be a result of lipofuscin pigment accum~ation. The inferior olive nucleus is also reported capable of mantling its rno~holo~~~ integrity into advanced age, without significant change in nerve cell number [23,24]. The physiological aging process in inferior olive must be, therefore, examined from another point of view. Zeman [35] reported that a stunting of the dendritic apparatus occurs when the rate of pigment production is higher than the rate of regenerative molecular synthesis. Several quantitative morphometrical studies have been carried out on the changes in cell processes in the neuropil with aging [6, 7, 9, 10, 19, 29, 30, 331. These studies reported atrophic or dystrophic changes in cell processes, or proliferation of astrogiiai cell processes. As to the inferior olive, quantitative uItr~t~ctur~ studies on the neuropil of aged mammals are very few. The present study was undertaken to assess the age-related ultrastructural changes in the neuropil in the rat inferior olive nucleus. METHOD

The animals used were male Wistar King rats. They were reared in closed colony with water and a standard laboratory rat chow. Four animals from each of the following ages were prepared: 3,6, 12,18,24 and 30 months old. Animals exhibiting any grossly detectable pathology at the time of perfusion

Copyright

Q 1982 ANKHO

Intemation~

Neuropil

were not used. The animals were anesthetized with pentobarbital sodium (35 mg/kg IP) and perfused through the heart with buffered saline followed by 4% paraformaldehyde in Millonig buffer at pH 7.3. Blocks of the brain stem were cut from the level of C, to about 0.3 cm rostral to the pontine-medulhuy junction, sliced and postfixed in 1% osmium tetraoxide, dehyd~ted and embedded in epon 812. Ultrathin sections were cut and stained with many1 acetate and lead citrate. The inferior olive nucleus was identified in 1 Frn thick toluidine blue stained sections (Fig. 1) from which areas were selected for ultrastructural examinations. An electron microscope (JEOL-100-B) was used to examine and photograph areas within the neuropil of the inferior olive at a direct rn~~~tion of 10,000~. Random samphngs within the neuropil were performed on two to three different tissue blocks from each rat producing 40 electron micrographs per subject animal, totalhng 960 electron micrographs. Photographic enlargement resulted in a final magnification of 30,000x on the prints. The profiles of axon terminals, dendrites and astrogliai processes in neuropil of each rni~r~~phs were traced. The percentages of the profiled areas in relation to the area of neuropil (named relative volume fraction) were then calculated using the image analyzer system. The “neuropil” in this context means the remainder of the inferior olive nucleus after exclusion of cell bodies, myelinated fibers and blood vessels. The criteria for identification of axon terminals were the presence of synaptic vesicles and/or of synaptic membrane differentiation. The criteria for identification of dendrites were as follows: any round or cylindrical profiles of cell processes which showed postsynaptic membrane differentiation on the surface and/or

Inc .-0361-9230/82/~381-~$03.~/0

382

IKARl,

HAYASNl

AND OGATA

any round or cylindrical profiles of greater than 0.3 pm in diameter which contained evenly spaced microtubl~s~ or any round or cylindrical profiles of smaller than 0.3 pm in diameter which contained no microtubles or glio~l~ent (the smallest dendrite and spine) 1181 and showed rather clear cytoplasmic matrix. The criteria for identification of astroglial cell processes were as follows: round, cylindri~ai or irregumrly shaped profibs which filled up the space between neuronal cell processes. These showed clear cytoplasmic matrix and contained contents, glycogen particles, and were poor in other organelIes [2, 12, IS]. The profIles which could not be ident~ed by the above mentioned criteria remained as unclassified. All of the jdent~able profiles except for unmyel~ted axons were traced and the percentage of the area of each profile for the area of neuropil (named relative volume fraction) was measured (Figs. 2,3,4). Figure 2 is a line drawing of the profiles of neuronal and astroglial processes in the ~e~ropil ~hoto~aphed in Fig. 4. Additional electron microscopic observations were made on the lipofuscin granules in the cell soma for one or two animals at 5 weeks, 3, 6, 18 and 30 months of age. FIG. I. inferior olive nucleus of a 3 month old rat. Epon 1 t.lm, toluidine blue stained. P: pyramis meduliae oblongatae. medz n. olivaris accesorius medialis. inf: n. olivaris inferior. dors: n. olivaris accessorius dorsalis. 50x

r

RESULTS

TabIe 1 shows the relative volume fractions of cell processes within the neuropil at various ages. Mean values (percentage) for each animal were derived from analyses of 40 electron micrographs. Mean values for groups of each age were calcuiated from ~dividual mean values and treated statistica~y with the ~de~ndent f-test for multiple comparison test (Dunnet’s test). The volume fraction of axon termi-

(B)

FIG.2.An example of profiles tracing the (A) presynaptic terminals and dendrites, (B) astroglial processes ofthe same rn~c~~~h

as in Fig. 4.

NEUROPIL

IN THE RAT INFERIOR

383

OLIVE TABLE

AGE-RELATED

CHANGES

I

IN THE RELATIVE VOLUME FRACTION INFERIOR OLIVE NUCLEUS

24

30

3

6

Mean _CS.D. of Terminal per Rat

RI R2 R3 R4

28rt 30t 29-c 28%

9 9 9 8

282 282 292 282

9 7 9 8

28% 272 25+ 292

8 8 7 9

28 27 28 28

292

9

28-+

8

272

8

28 rt 9

27 + 7*

24 r 7*

28 25 26 28

28 28 25 29

25 26 24 29

Mean rt SD. of Dendrite per Rat

RI R2 R3 R4

Mean rt S.D. per Group Mean rt SD. of Glial Process per Rat Mean t S.D. per Group Unclassified and other axon profiles except for axon

RI R2 R3 R4

32rt 8 34Icr: 11 34 * 10 32-c 8

33 -c 31+ 31-c 312

10 9 6 8

32~

8

33%

6rt 6r: 6-t 6r

3 3 4 3

6rt

3

34

18

IN THE

Age (month)

Mean r S.D. per Group

12

(410)OF NEUROPIL

29k 32 f 34 -c 282

8 10 10 7

9

31+-

9

7F 7* 6+ 7k

3 4 4 4

7k

4

32

r 9 -t- 8 r. 8 f 9

r * it rt

8 7 8 9

23 26 26 22

r f k f

i + + +

6 7 8 7

8 9 7 7

24 23 22 25

+ 7 _’ 6 -e 6 2 8

+ 2 2 +

8 9 7 8

27 +- 8*

28 + 8*

26 r 8*

10-i- 5 9+ 4 11-t 5 9t 4

13 rt 5 954 13 + 5 12 t 5

11 14 10 13

15 + 4 17 z!z5 17 t 4 16 -t 4

lo*

12 c 5*

12 r 5*

16 t 4*

34

36

34

5 32

25 1?16 k 4 k 4

terminals n=160. *p
nals tends to decrease over the lifespan under study. Compared with 3 month-old rats, the volume fraction of axon terminals at the ages of 24 and 30 months significantly decreased (p
amount of lipofnscin granules are observed. The degree of accum~ation of the lipofuscin granules seemed to increase with aging. Figure 6 shows a neuronal cell body from the inferior olive nucleus at the age of 24 months in which large mass of lipofuscin granules are observed. As far as we observed, there was no tendency to increase dark cells or other degenerative nerve cells in the inferior olive nucleus of 30 month old rats. DISCUSSION

In the present study, we observed relative decrease in the areas of axon terminals and dendrites, and relative increase in the area of astroglial processes in the inferior olive nucleus of the senescent rat. These findings may represent the common changes in the aging process of neuronal tissue. It seems reasonable to suggest that the decrease in the neuronal cell processes and the increase in the astroglial processes in the inferior olive nucleus with advancing age are the glio-neuronal or neuro-glial age-related changes which indicate the gradual reduction in the cellular efficiency. Concerning the axon terminals, a few age-related changes have been reported. They were the decrease in synaptic density [8, 9, 111, decrease in the terminal area [8,16] and synaptic vesicles depletion [I 1,161. Fujisawa and Shiraki [8] reported the slow progressive decrease in the synaptic density and the relative volume fraction of axon terminals in the gracile nucleus of aged rats. Geinisman and Bondareff [9]

IKARI, HAYASHI

AND OCiATA

FIG. 3. An example of neuropil of the inferior olive nudeus, 3 month old rat, with presynaptic terminals fT), dendrites (D) and as&o&al processes (G). Small amount of astroglial processes are observed. M: myelin sheath. 30,ooox

NEUROPIL

IN THE

RAT INFERIOR

OLIVE

FIG. 4. An example ofneuropil of the inferior olive nucieus, 30 month ofd rat, with presynaptic terminals (T), dendrites (D) and astroglial processes (G). Note the increase of astroglial profiles which shows clear cytoplasmic matrix containing glycogen particles (gly) and glial filaments (fil). Arrows indicate gap junctions between adjacent astrocytic processes. 30$00x

386

IKARI,

HAYASHI

AND OGATA

FIG. 5. An electron micrograph of a neuronal cell body of a rat inferior olive nucleus, 5 weeks old. Small amount of lipofuscin granules are observed. N: nucleus, L: lipofuscin granules.

reported a loss of synapses in the molecular layer of the dentate gyrus in aged rats. Hassan and Glees [ll] in their study of age changes in the hippocampus noted that while axo-somatic contacts were a prominent feature of younger rats, these decreased in aged rats along with characteristic membrane thickening and accumulution of synaptic vesicles. Landfield et al. [16] also reported reduced numbers of synaptic vesicles in the terminals and a trend toward reduced terminal size of aged rat hippocampus. Neurochemical studies reported that changes in the acetylcholinesterase activity, norepinephline, serotonine and GABA contents in the brain have been associated with increasing age [22,28]. These findings may suggest reduced functioning of synapse with aging. One possible mechanism for the axonal atrophy and reduction of synaptic activity which accompanies advancing age is a “dying-back” degeneration (or retrograde axonal degeneration) of neurons. This could be explained by the perikaryal insufficiency or axonal transport system damage. Namely, there is a symmetrical axonal degeneration, beginning distally in vulnerable regions of the nervous system, spreading proximally, and sometimes involving the neuron cell body [IS, 25, 26, 321. Bondareff et al. reported age-related changes in axonal transport of glycoprotein in the rat septodentate pathway [I]. Preceeding the neuronal cell loss, the atrophic changes

may occur in the axon terminals by just such a very slow “dying-back” process of aging. some other studies have shown constant synaptic density throughout the adult life (ages 16-72 years) [ 131and at the age of 89 years [5] in human cortex. Any speculation, therefore, must be made carefully. Concerning the dendrites, age-related dendritic atrophy has been reported from the investigations of Golgi preparations [6, 7, 29, 30, 33, 341 and electron microscopic studies [7, 8, lo]. By modifications of Golgi methods, age-related changes in the dendritic domen of neocortical [29] and archicortical [30] neurons in human materials were characterized by patchy loss of dendritic spines, progressive decrement of dendritic systems and subsequent degeneration of apical shaft with final loss of the cell body. Some experimental studies in Golgi preparations [6, 7, 33, 341 and quantitative electron microscopic analyses [7,8, IO] also reported that atrophic changes affect more peripheral portions of neuronal dendritic tree, especially spines. The age-related dendritic atrophy could be understood as the result of partial detierentation which occurs in axo-spinous synapses [ 10,291. Concerning the astroglial processes, several studies reported an increase in the number of their profiles or of their volume in the dentate gyrus [I 1,161, glacile nucleus of the senescent rats [8], and cerebral cortex of the senescent

NEUROPLL IN THE RAT INFERIOR

OLIVE

FIG. 6. An electron micrograpb showing a neuronal cell body of a rat inferior olive nucleus, 30 months otd. Large mass of lipofuscin granules are observed. N: nucleus, L: lipofuscin granules.

human brain 143. However there were no sign~~ant aherations in astrc ~ytic cell soma [4,11]. Terminals showing early processes of degenerative alterations are often seen in proximity to reac :tive astrocytic processes [Hi]. Glial cells have characteristic J membrane processes for amino acid transport which can in aeract with the microenvironment. During glial proliferation or gliosis which can cause disturbance of the

micr~nvironment and ne~on~ function, selective increase in amino acid uptake may occur [ 171. The possible f‘unctions of astrocytes are as follows: (I) insulation of unm! yrelinated neuronal processes fkom each other, (2) isolat :ion and removal of degenerative debris, (3) a channel for * moving metabolites from blood vessels to neurons, (4) regu Iation of synaptic activity. Thus, there is a cross relation between

388

IKARI. HAYASHI

AND OGATA

neuron and astroglia [14]. Ravens 1271 insisted on the So far as the physiological aging process is concerned, it angio-Rio-ne~on~ unit theory and that the pathological seems reasonable to suggest that the mo~hologica1 manifeschanges of any one of these three elements resulted in subtations are the accumulation of Iipofusein granules in ceU sequent pathological changes of the rest of the two elements. soma, atrophy of neuronal celI processes and proliferation of The relationships of the intracellular lipofuscin concenastroglial processes in neuropil. It is therefore necessary to tration with the reduction of cell number of cellular efficlarify the relationships between the lipofuscin concentraciency in aged rat have not been clear [23, 24, 311. In the tion in nerve cells and the changes in the neuropil. present study, we observed the progressive accumulation of the lipofuscin granules with advancing age in the neuronal cell bodies. We did not count the nerve cell numbers, so that ACKNOWLEDGEMENTS we can not comment on the change in nerve cell population in the inferior olive. There was however, no tendency to We should like to thank Prof. Hiroyuki Nakao and Prof. Yutaka increase dark cells or other degenerative nerve cells in aged Oomura for important suggestions, Ms. Junko Ono for technical assistance and Ms. Yoko Kondo for expert secretarial help. rats. REFERENCES I. Bondareff,

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