RAPID COMMUNICATION
DISPERSION
OF
CISTERNAE
I N AGING CNS NEURONS:
ZIPGRAM
OF
ROUGH ENDOPLASMIC RETICULUM
A STRICTLY LINEAR TREND
JAMES W. H I N D S AND NANCY A. MCNELLY Department o f Anatomy, Boston U n i v e r s i t y School o f Medicine, Boston, Massachusetts 02118 ABSTRACT D i s p e r s i o n o f c i s t e r n a e o f rough endoplasmic r e t i c u l u m (RER) i n a g i n g r a t s has been determined q u a n t i t a t i v e l y f o r m i t r a l c e l l s o f t h e o l f a c t o r y b u l b and P u r k i n j e c e l l s o f t h e c e r e b e l l u m u s i n g a r e c e n t l y p u b l i s h e d morphometric t e c h n i q u e (Cruz Orive, ' 7 6 ) . I n b o t h c e l l types d i s p e r s i o n o f c i s t e r n a e o c c u r r e d t h r o u g h o u t t h e p e r i o d s t u d i e d and i n a l i n e a r f a s h i o n . The observed RER d i s p e r s i o n cannot be a t t r i b u t e d t o a decrease i n t h e t o t a l amount o f RER, s i n c e t h e t o t a l amount o f RER was c o n s t a n t i n P u r k i n j e c e l l s and i n c r e a s e d i n m i t r a l c e l l s d u r i n g t h e ages s t u d i e d . U n t i l r e c e n t l y t h e r e was no easy way t o q u a n t i f y changes i n p a t t e r n o f c e l l u l a r o r g a n e l l e s which occur as a r e s u l t o f experimental procedures o r d u r i n g development and aging. Orive,
'76).
Now, however, such a method i s a v a i l a b l e (Cruz
We have used t h i s method t o s t u d y t h e p a t t e r n o f rough endo-
plasmic r e t i c u l u m (RER) i n a g i n g CNS neurons. (Hasan and Glees,
'73; Johnson and Miquel,
I t had p r e v i o u s l y been n o t e d
'74; and Sekhon and Maxwell,
'74)
t h a t t h e h i g h l y ordered p a r a l l e l c i s t e r n a e o f RER c h a r a c t e r i s t i c o f young neurons seem t o become d i s p e r s e d d u r i n g aging. nomenon q u a n t i t a t i v e l y .
We have now s t u d i e d t h i s phe-
Two l a r g e and w e l l - d e f i n e d neuronal c e l l t y p e s have
been examined, m i t r a l c e l l s o f t h e o l f a c t o r y b u l b and c e r e b e l l a r P u r k i n j e cells. MATERIALS AND METHODS
For t h e p r e s e n t s t u d y a t o t a l o f 16 r a t s was used:
two male Sprague-Dawley r a t s a t each age (3, 12, 24, and 27 months) f o r m i t r a l
43 3
c e l l measurements and two male specific-pathogen-free, Fisher 344 r a t s (Charles River Breeding Labs) a t each age ( 4 , 12, 18, and 24 months) f o r Purkinje c e l l measurements. Fixation and embedding of t i s s u e s were carried out a s previously described (Hinds and McNelly, ' 7 7 ) .
For mitral c e l l measurements, 70-nm sec-
t i o n s were taken in the mid-lateral portion of the r i g h t o l f a c t o r y bulb, 1000 pm
from the rostra1 t i p of the glomerular layer.
Cells from t h i s region were
chosen because they were close t o the mean s i z e f o r mitral c e l l s in the olfactory bulb (Hinds and McNelly, unpublished).
I n order t o obtain a random sam-
ple of c e l l sections, micrographs of a l l rnitral c e l l s in the section, both those with nuclei and those without, were taken using an AEI Corinth 275 elec-
t r o n microscope.
The number o f c e l l s per section varied from 19 t o 39 in d i f -
f e r e n t animals.
For Purkinje c e l l measurements 70-nm s a g i t t a l sections of nod-
ulus near t h e midline were cut and, in the same way a s f o r mitral c e l l s , micrographs of a t o t a l of 20 Purkinje c e l l s (10 from e i t h e r s i d e of the t i p of the nodulus) were taken from each animal.
Photographs of mitral and Purkinje
c e l l s were printed a t a f i n a l magnification of 10,000 X. A measure o f the dispersion o f RER c i s t e r n a e was obtained with the method
of Cruz Orive ( ' 7 6 )
A rectangular a r r a y of open c i r c l e s , with t h e i r centers
1.5 cm a p a r t , was p aced over the electron micrographs, and t h e number of c i r c l e s intersecting 1 2 , or 3 o r more d i f f e r e n t RER p r o f i l e s was recorded
(N1, N2, N3+).
For mitral c e l l s c i r c l e s with a diameter o f 5.5 mm were chosen
and f o r Purkinje c e l l s c i r c l e s 7.0 mm in diameter, following t h e procedure outlined in Cruz Orive ( ' 7 6 ) .
The measure t h a t we used f o r dispersion was t h e
f r a c t i o n of c i r c l e s i n t e r s e c t i n g two o r more p r o f i l e s o f RER: Q+
= (N2
+ N3+)
/ Ntotal,
f o r each animal.
Thus i f c i s t e r n a e of RER were more
dispersed in a given animal, a smaller ii2t would be expected.
A measure of
TABLE 1 Olfactory Bulb 12 mo. 24 mo. 27 mo. 3 mo. **cell volume (pm3) 151m.5) 19-2) 288-0) 423-5) *RER s u r f a c e ( urn2) 827( 585.0) 1129( 5155) 1570( 521.5) 2391( 553.0) SV (urn-1) 0.786(5. 040) 0.818( i .044) 0.768(5. 038) 0.735( 5 .026) Cerebel 1um 4 mo. 12 mo. 18 mo. 24 rno. c e l l volume (um3) 406m6) 4 1 0 m ) 462-1 4202(+181) 1334(+99.5) 1407(i38.0) RER s u r f a c e ( p m 2 ) 1155(+18.5) 1512(+151) 0.334(+. 038) 0.426( 5 .062) 0.376(5. 023) 0.351( 5 .010) s (pm-1) 6.02(+.065) 5.81(+.190) 6.11(+.270) 5.89(+.130) P?L l e n g t h (cm) * P < O . O l ; **P< KO31 means 5 standard e r r o r of mean; analyses of variance:
the s u r f a c e a r e a of RER membranes per u n i t volume of perikaryal cytoplasm ( S v ) can a l s o be obtained from the f i g u r e s used t o measure d i s p e r s i o n (Cruz
Orive, ' 7 6 ) .
Sv was m u l t i p l i e d by the mean perikaryal volume f o r each animal
t o determine the mean t o t a l s u r f a c e a r e a of RER per c e l l f o r each animal. Perikaryal volume was found by determining, on 1-um s e c t i o n s a d j a c e n t t o the t h i n s e c t i o n s used f o r morphometry, the mean s i z e of Purkinje and m i t r a l c e l l nuclei and s u b t r a c t i n g t h i s f i g u r e from the mean Purkinje and m i t r a l c e l l body s i z e i n the manner described in a previous paper (Hinds and McNelly, ' 7 7 ) . For m i t r a l c e l l s 90 c e l l s from three d i f f e r e n t rostrocaudal l e v e l s were measured i n each animal; f o r Purkinje c e l l s 50 c e l l s from each nodulus were measured.
The l e n g t h of the Purkinje c e l l l a y e r was determined w i t h a map measurer on camera l u c i d a drawings of 1-pm s a g i t t a l s e c t i o n s of the entire vermis i n o r d e r t o o b t a i n a measure of c e r e b e l l a r s i z e w i t h age. S t a t i s t i c a l tests were taken from Sokal and Rohlf ( ' 6 9 ) .
Bartlett's
t e s t f o r homogeneity of variances was done f o r a l l d a t a f o r which analyses o f v a r i a n c e were performed, and i n a l l c a s e s i t was non-significant. RESULTS
D u r i n g aging, RER c i s t e r n a e dispersed i n a l i n e a r fashion i n
both m i t r a l c e l l s and Purkinje c e l l s ( f i g s . 1-3).
43 5
That both should show a
s i m i l a r increased d i s p e r s i o n i s perhaps s u r p r i s i n g , s i n c e t h e t o t a l amount o f
RER per c e l l i s i n c r e a s i n g g r e a t l y i n m i t r a l c e l l s , w h i l e i t i s a p p a r e n t l y constant i n P u r k i n j e c e l l s ( t a b l e 1).
I n m i t r a l c e l l s t h i s increase r e f l e c t s
t h e l a r g e i n c r e a s e i n t h e volume o f m i t r a l c e l l bodies ( t a b l e 1 ) a s s o c i a t e d w i t h t h e o v e r a l l i n c r e a s e i n t h e s i z e o f t h e o l f a c t o r y b u l b d u r i n g most o f a d u l t l i f e (Hinds and McNelly, '77).
I n t h e c e r e b e l l u m volumes o f P u r k i n j e
c e l l bodies do n o t change w i t h age and n e i t h e r does t h e l e n g t h o f t h e P u r k i n j e c e l l l a y e r (PCL) i n a s e c t i o n of e n t i r e vermis ( t a b l e 1). D e s p i t e t h e f a c t t h a t RER c i s t e r n a e show i n c r e a s i n g d i s p e r s i o n w i t h age, measurements o f t h e surface-to-volume r a t i o ( s u r f a c e o f RER c i s t e r n a e t o volume o f p e r i karyon) have shown no s i g n i f i c a n t change ( t a b l e 1 ) . DISCUSSION
R e s u l t s o f t h e p r e s e n t s t u d y show t h a t RER c i s t e r n a e i n
neurons o f two d i f f e r e n t r e g i o n s o f t h e CNS become, on t h e average, l e s s c l o s e l y spaced d u r i n g a d u l t l i f e .
T h i s decrease i n average p r o x i m i t y o f one
c i s t e r n o f RER t o another cannot be a t t r i b u t e d t o a general decrease i n t h e amount o f RER s i n c e t h e volume f r a c t i o n o f RER i n b o t h m i t r a l and P u r k i n j e c e l l s remains r e l a t i v e l y c o n s t a n t t h r o u g h o u t t h e age p e r i o d s t u d i e d .
Fur-
thermore, t h e t o t a l amount o f RER was c o n s t a n t f o r P u r k i n j e c e l l s and i n creased i n m i t r a l c e l l s .
Thus we conclude t h a t d u r i n g a g i n g t h e r e probably
i s a r e a l d i s p e r s a l o f c i s t e r n a e o f RER from t h e ordered s t a t e o f p a r a l l e l c i s t e r n a e found i n these neurons i n young animals.
FIGURE LEGENDS 1 Tracings o f m i t r a l c e l l s c l o s e t o t h e mean RER d i s p e r s i o n f o r 3 ( l e f t ) and 27 ( r i g h t ) months. 4,000 X. 2 Measure o f t h e closeness o f m i t r a l c e l l RER c i s t e r n a e t o one another ( B +) as a f u n c t i o n o f age. Presence o f r e g r e s s i o n : F(1,2)=6,442.2, P=6.00016; a n a l y s i s o f variance: F(3,4)=25.9, P=0.0044. 3 BZ+ o f P u r k i n j e c e l l RER as a f u n c t i o n of age. Presence o f r e g r e s s i o n : F( 1,2)=126.0, P=0.0078; a n a l y s i s of v a r i a n c e : F( 3,4)=51.7, P=0. 0012.
436
40
41
%
70
20
2
p2+ 01
1
5
p2+ of R.E.R.of PURKINJE CELLS
R.E.R. of MITRAL CELLS
10
15
AGE (months)
I
I
20
25
4
I0
1;
AGE (months)
io
25
The most surprising finding of t h e present study i s t h a t in both regions studied there occurs a highly l i n e a r decrease in t h e measured closeness of RER cisternae throughout a d u l t l i f e .
The only other c e l l organelle we a r e
aware of t h a t has been reported t o show l i n e a r changes with age i s lipofuscin ( S t r e h l e r e t a l . , '59; Samorajsk in preparation).
e t a1 ., '68; Hinds and McNelly, manuscript
In addition, 1 pid peroxides in t h e brains of r a t s have
been reported t o increase l i n e a r y during l i f e (Yoshikawa and Hirai, '67). Since RER membranes a r e t h o u g h t t o be a potential source of lipofuscin by peroxidation of t h e i r unsaturated l i p i d s and subsequent cross-linking with proteinaceous components (Tappel , '65; Packer e t a l . , ' 6 7 ) , i t i s possible t h a t a l i n e a r change in l i p i d peroxidation of RER membranes with age may be in some way responsible f o r b o t h t h e observed dispersion of c i s t e r n a e seen in t h e present study and t h e increase in lipofuscin. ACKNOWLEDGMENTS
We a r e grateful t o Doctor D. W. Vaughan f o r her expert
perfusion o f t h e animals used in t h i s study and her c r i t i c a l reading of the manuscript.
Supported by USPHS Grant 9 PO1 AG 00001.
LITERATURE CITED
Cruz Orive, L-M. 1976 Quantifying ' p a t t e r n ' : A stereological approach. J . Microsc., 107: 1-18. Hasan, M . , and P . Glees 1973 Ultrastructural age changes in hippocampa1 neurons, synapses and neuroglia. Exp. Gerontol., 8: 75-83. Hinds, J . W . , and N . A. McNelly 1977 Aging of t h e r a t olfactory bulb: Growth and atrophy of c o n s t i t u e n t layers and changes in t h e s i z e and number of mitral c e l l s . J . Comp. Neur., 171: 345-368. Johnson, J . E . , J r . , and J . Miquel 1974 Fine s t r u c t u r a l changes in t h e l a t e r a l v e s t i b u l a r nucleus of aging r a t s . Mech. Aging Dev., 3: 203-224. Packer, L., D. W. Deamer and R. L . Heath 1967 Regulation and deterioration of s t r u c t u r e in membranes. Adv. Gerontol. Res., 2 : 77-120. Samorajski, T . , J . M. Ordy and P. Rady-Reimer 1968 Lipofuscin pigment accumulation in the nervous system of aging mice. Anat. Rec., 160: 555-574. Sekhon, S . S . , and D. S. Maxwell 1974 Ultrastructural changes in neurons of the spinal a n t e r i o r horn of aging mice with p a r t i c u l a r reference t o t h e accumulation of lipofuscin pigment. J . Neurocytol., 3 : 59-72.
438
Sokal, R. R., and F. J. Rohlf 1969 Biometry. W. H. Freeman and Co., San Francisco. Strehler, B. L., D. D. Mark, A . S. Mildvan and M. V. McGee 1959 Rate and magnitude of age pigment accumulation in the human myocardium. J. Gerontol., 14: 430-439. Tappel, A. L. 1965 Free-radical lipid peroxidation damage and its inhibition by vitamin E and selenium. Fed. Proc., 24: 73-78. Yoshikawa, M., and S. Hirai 1967 Lipid peroxide formation in the brain of aging rats. J. Gerontol. , 22: 162-165.
439
DISPERSION
OF
CISTERNAE
I N AGING CNS NEURONS:
ZIPGRAM
OF
ROUGH ENDOPLASMIC RETICULUM
A STRICTLY LINEAR TREND
JAMES W. H I N D S AND NANCY A. MCNELLY Department o f Anatomy, Boston U n i v e r s i t y School o f Medicine, Boston, Massachusetts 02118 ABSTRACT D i s p e r s i o n o f c i s t e r n a e o f rough endoplasmic r e t i c u l u m (RER) i n a g i n g r a t s has been determined q u a n t i t a t i v e l y f o r m i t r a l c e l l s o f t h e o l f a c t o r y b u l b and P u r k i n j e c e l l s o f t h e c e r e b e l l u m u s i n g a r e c e n t l y p u b l i s h e d morphometric t e c h n i q u e (Cruz Orive, ' 7 6 ) . I n b o t h c e l l types d i s p e r s i o n o f c i s t e r n a e o c c u r r e d t h r o u g h o u t t h e p e r i o d s t u d i e d and i n a l i n e a r f a s h i o n . The observed RER d i s p e r s i o n cannot be a t t r i b u t e d t o a decrease i n t h e t o t a l amount o f RER, s i n c e t h e t o t a l amount o f RER was c o n s t a n t i n P u r k i n j e c e l l s and i n c r e a s e d i n m i t r a l c e l l s d u r i n g t h e ages s t u d i e d . U n t i l r e c e n t l y t h e r e was no easy way t o q u a n t i f y changes i n p a t t e r n o f c e l l u l a r o r g a n e l l e s which occur as a r e s u l t o f experimental procedures o r d u r i n g development and aging. Orive,
'76).
Now, however, such a method i s a v a i l a b l e (Cruz
We have used t h i s method t o s t u d y t h e p a t t e r n o f rough endo-
plasmic r e t i c u l u m (RER) i n a g i n g CNS neurons. (Hasan and Glees,
'73; Johnson and Miquel,
I t had p r e v i o u s l y been n o t e d
'74; and Sekhon and Maxwell,
'74)
t h a t t h e h i g h l y ordered p a r a l l e l c i s t e r n a e o f RER c h a r a c t e r i s t i c o f young neurons seem t o become d i s p e r s e d d u r i n g aging. nomenon q u a n t i t a t i v e l y .
We have now s t u d i e d t h i s phe-
Two l a r g e and w e l l - d e f i n e d neuronal c e l l t y p e s have
been examined, m i t r a l c e l l s o f t h e o l f a c t o r y b u l b and c e r e b e l l a r P u r k i n j e cells. MATERIALS AND METHODS
For t h e p r e s e n t s t u d y a t o t a l o f 16 r a t s was used:
two male Sprague-Dawley r a t s a t each age (3, 12, 24, and 27 months) f o r m i t r a l
43 3
c e l l measurements and two male specific-pathogen-free, Fisher 344 r a t s (Charles River Breeding Labs) a t each age ( 4 , 12, 18, and 24 months) f o r Purkinje c e l l measurements. Fixation and embedding of t i s s u e s were carried out a s previously described (Hinds and McNelly, ' 7 7 ) .
For mitral c e l l measurements, 70-nm sec-
t i o n s were taken in the mid-lateral portion of the r i g h t o l f a c t o r y bulb, 1000 pm
from the rostra1 t i p of the glomerular layer.
Cells from t h i s region were
chosen because they were close t o the mean s i z e f o r mitral c e l l s in the olfactory bulb (Hinds and McNelly, unpublished).
I n order t o obtain a random sam-
ple of c e l l sections, micrographs of a l l rnitral c e l l s in the section, both those with nuclei and those without, were taken using an AEI Corinth 275 elec-
t r o n microscope.
The number o f c e l l s per section varied from 19 t o 39 in d i f -
f e r e n t animals.
For Purkinje c e l l measurements 70-nm s a g i t t a l sections of nod-
ulus near t h e midline were cut and, in the same way a s f o r mitral c e l l s , micrographs of a t o t a l of 20 Purkinje c e l l s (10 from e i t h e r s i d e of the t i p of the nodulus) were taken from each animal.
Photographs of mitral and Purkinje
c e l l s were printed a t a f i n a l magnification of 10,000 X. A measure o f the dispersion o f RER c i s t e r n a e was obtained with the method
of Cruz Orive ( ' 7 6 )
A rectangular a r r a y of open c i r c l e s , with t h e i r centers
1.5 cm a p a r t , was p aced over the electron micrographs, and t h e number of c i r c l e s intersecting 1 2 , or 3 o r more d i f f e r e n t RER p r o f i l e s was recorded
(N1, N2, N3+).
For mitral c e l l s c i r c l e s with a diameter o f 5.5 mm were chosen
and f o r Purkinje c e l l s c i r c l e s 7.0 mm in diameter, following t h e procedure outlined in Cruz Orive ( ' 7 6 ) .
The measure t h a t we used f o r dispersion was t h e
f r a c t i o n of c i r c l e s i n t e r s e c t i n g two o r more p r o f i l e s o f RER: Q+
= (N2
+ N3+)
/ Ntotal,
f o r each animal.
Thus i f c i s t e r n a e of RER were more
dispersed in a given animal, a smaller ii2t would be expected.
A measure of
TABLE 1 Olfactory Bulb 12 mo. 24 mo. 27 mo. 3 mo. **cell volume (pm3) 151m.5) 19-2) 288-0) 423-5) *RER s u r f a c e ( urn2) 827( 585.0) 1129( 5155) 1570( 521.5) 2391( 553.0) SV (urn-1) 0.786(5. 040) 0.818( i .044) 0.768(5. 038) 0.735( 5 .026) Cerebel 1um 4 mo. 12 mo. 18 mo. 24 rno. c e l l volume (um3) 406m6) 4 1 0 m ) 462-1 4202(+181) 1334(+99.5) 1407(i38.0) RER s u r f a c e ( p m 2 ) 1155(+18.5) 1512(+151) 0.334(+. 038) 0.426( 5 .062) 0.376(5. 023) 0.351( 5 .010) s (pm-1) 6.02(+.065) 5.81(+.190) 6.11(+.270) 5.89(+.130) P?L l e n g t h (cm) * P < O . O l ; **P< KO31 means 5 standard e r r o r of mean; analyses of variance:
the s u r f a c e a r e a of RER membranes per u n i t volume of perikaryal cytoplasm ( S v ) can a l s o be obtained from the f i g u r e s used t o measure d i s p e r s i o n (Cruz
Orive, ' 7 6 ) .
Sv was m u l t i p l i e d by the mean perikaryal volume f o r each animal
t o determine the mean t o t a l s u r f a c e a r e a of RER per c e l l f o r each animal. Perikaryal volume was found by determining, on 1-um s e c t i o n s a d j a c e n t t o the t h i n s e c t i o n s used f o r morphometry, the mean s i z e of Purkinje and m i t r a l c e l l nuclei and s u b t r a c t i n g t h i s f i g u r e from the mean Purkinje and m i t r a l c e l l body s i z e i n the manner described in a previous paper (Hinds and McNelly, ' 7 7 ) . For m i t r a l c e l l s 90 c e l l s from three d i f f e r e n t rostrocaudal l e v e l s were measured i n each animal; f o r Purkinje c e l l s 50 c e l l s from each nodulus were measured.
The l e n g t h of the Purkinje c e l l l a y e r was determined w i t h a map measurer on camera l u c i d a drawings of 1-pm s a g i t t a l s e c t i o n s of the entire vermis i n o r d e r t o o b t a i n a measure of c e r e b e l l a r s i z e w i t h age. S t a t i s t i c a l tests were taken from Sokal and Rohlf ( ' 6 9 ) .
Bartlett's
t e s t f o r homogeneity of variances was done f o r a l l d a t a f o r which analyses o f v a r i a n c e were performed, and i n a l l c a s e s i t was non-significant. RESULTS
D u r i n g aging, RER c i s t e r n a e dispersed i n a l i n e a r fashion i n
both m i t r a l c e l l s and Purkinje c e l l s ( f i g s . 1-3).
43 5
That both should show a
s i m i l a r increased d i s p e r s i o n i s perhaps s u r p r i s i n g , s i n c e t h e t o t a l amount o f
RER per c e l l i s i n c r e a s i n g g r e a t l y i n m i t r a l c e l l s , w h i l e i t i s a p p a r e n t l y constant i n P u r k i n j e c e l l s ( t a b l e 1).
I n m i t r a l c e l l s t h i s increase r e f l e c t s
t h e l a r g e i n c r e a s e i n t h e volume o f m i t r a l c e l l bodies ( t a b l e 1 ) a s s o c i a t e d w i t h t h e o v e r a l l i n c r e a s e i n t h e s i z e o f t h e o l f a c t o r y b u l b d u r i n g most o f a d u l t l i f e (Hinds and McNelly, '77).
I n t h e c e r e b e l l u m volumes o f P u r k i n j e
c e l l bodies do n o t change w i t h age and n e i t h e r does t h e l e n g t h o f t h e P u r k i n j e c e l l l a y e r (PCL) i n a s e c t i o n of e n t i r e vermis ( t a b l e 1). D e s p i t e t h e f a c t t h a t RER c i s t e r n a e show i n c r e a s i n g d i s p e r s i o n w i t h age, measurements o f t h e surface-to-volume r a t i o ( s u r f a c e o f RER c i s t e r n a e t o volume o f p e r i karyon) have shown no s i g n i f i c a n t change ( t a b l e 1 ) . DISCUSSION
R e s u l t s o f t h e p r e s e n t s t u d y show t h a t RER c i s t e r n a e i n
neurons o f two d i f f e r e n t r e g i o n s o f t h e CNS become, on t h e average, l e s s c l o s e l y spaced d u r i n g a d u l t l i f e .
T h i s decrease i n average p r o x i m i t y o f one
c i s t e r n o f RER t o another cannot be a t t r i b u t e d t o a general decrease i n t h e amount o f RER s i n c e t h e volume f r a c t i o n o f RER i n b o t h m i t r a l and P u r k i n j e c e l l s remains r e l a t i v e l y c o n s t a n t t h r o u g h o u t t h e age p e r i o d s t u d i e d .
Fur-
thermore, t h e t o t a l amount o f RER was c o n s t a n t f o r P u r k i n j e c e l l s and i n creased i n m i t r a l c e l l s .
Thus we conclude t h a t d u r i n g a g i n g t h e r e probably
i s a r e a l d i s p e r s a l o f c i s t e r n a e o f RER from t h e ordered s t a t e o f p a r a l l e l c i s t e r n a e found i n these neurons i n young animals.
FIGURE LEGENDS 1 Tracings o f m i t r a l c e l l s c l o s e t o t h e mean RER d i s p e r s i o n f o r 3 ( l e f t ) and 27 ( r i g h t ) months. 4,000 X. 2 Measure o f t h e closeness o f m i t r a l c e l l RER c i s t e r n a e t o one another ( B +) as a f u n c t i o n o f age. Presence o f r e g r e s s i o n : F(1,2)=6,442.2, P=6.00016; a n a l y s i s o f variance: F(3,4)=25.9, P=0.0044. 3 BZ+ o f P u r k i n j e c e l l RER as a f u n c t i o n of age. Presence o f r e g r e s s i o n : F( 1,2)=126.0, P=0.0078; a n a l y s i s of v a r i a n c e : F( 3,4)=51.7, P=0. 0012.
436
40
41
%
70
20
2
p2+ 01
1
5
p2+ of R.E.R.of PURKINJE CELLS
R.E.R. of MITRAL CELLS
10
15
AGE (months)
I
I
20
25
4
I0
1;
AGE (months)
io
25
The most surprising finding of t h e present study i s t h a t in both regions studied there occurs a highly l i n e a r decrease in t h e measured closeness of RER cisternae throughout a d u l t l i f e .
The only other c e l l organelle we a r e
aware of t h a t has been reported t o show l i n e a r changes with age i s lipofuscin ( S t r e h l e r e t a l . , '59; Samorajsk in preparation).
e t a1 ., '68; Hinds and McNelly, manuscript
In addition, 1 pid peroxides in t h e brains of r a t s have
been reported t o increase l i n e a r y during l i f e (Yoshikawa and Hirai, '67). Since RER membranes a r e t h o u g h t t o be a potential source of lipofuscin by peroxidation of t h e i r unsaturated l i p i d s and subsequent cross-linking with proteinaceous components (Tappel , '65; Packer e t a l . , ' 6 7 ) , i t i s possible t h a t a l i n e a r change in l i p i d peroxidation of RER membranes with age may be in some way responsible f o r b o t h t h e observed dispersion of c i s t e r n a e seen in t h e present study and t h e increase in lipofuscin. ACKNOWLEDGMENTS
We a r e grateful t o Doctor D. W. Vaughan f o r her expert
perfusion o f t h e animals used in t h i s study and her c r i t i c a l reading of the manuscript.
Supported by USPHS Grant 9 PO1 AG 00001.
LITERATURE CITED
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