In vivo brain calcium homeostasis during aging

In vivo brain calcium homeostasis during aging

Mechamsms of Ageing and Development, 37 (1986) 1-12 1 Elsevier Scientific Pubhshers Ireland Ltd IN VIVO BRAIN CALCIUM HOMEOSTASIS DURING AGING GAR...

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Mechamsms of Ageing and Development, 37 (1986) 1-12


Elsevier Scientific Pubhshers Ireland Ltd


GARY GIBSON, PETER PERRINO and GERALD A DIENEL* Cornell University Medtcal College, Burke Rehabdttatton Center, 785 Mamaroneck Avenue, Whtte Plams, N Y 10605 (U S A )

(Recewed November 15th, 1985) (Revision received August 2rid, 1986)

SUMMARY Since m vttro experiments suggest that brain calcium metabohsm is altered wlth aging, the estimated rate of calcmm uptake by the brain m vtvo was determined with senescence Calcmm-45 incorporation mto cortex, strlatum, luppocampus, cerebellum, forebram, rnldbram and bramstem was determmed in 3-, 10- and 30-month-old ml~e at 5 h after either an intravenous or mtrapentoneal rejection Calcium uptake (brain dpm/mg protein &vlded by blood specific activity at 5 h) into these regions dechned 19-33% at 10 months and 41-51% at 30 months Subcellular fracuonatlon of the cortex revealed that the decrease was smular m P~ (myehn, nuclel and tissue debris), P2 (synaptosomes, mltochondna and myehn) and $2 (mlcrosomes, ribosomes and cytosol) Brain calcmm concentraUons dechned with age m brain stem (-62%) and mldbram (--48%) but &d not slgmficantly vary with age m the other regions These results support the suggestion that alterations m calcium homeostasis may underhe age-related changes m neurotransmltter metabohsm

Key words Calcium, Aging, Brain

INTRODUCTION Several hnes of evldence suggest that altered calcmm metabohsm underhes age-related deficits m neuronal function Calcmm-dependent release of acetylchohne dlmlmshes w~th *Present address Laboratory of Cerebral Metabohsm, National Institute of Mental Health, Bethesda, MD 20892, U S A Abbreviations TES, N-trls[hydroxymethyl]methyl-2-ammo ethane sulfomc acid, EGTA, ethylene (bls-beta ammoethylene ether) N,N,N',N'-tetraacetlc acad

0047-6374/86/$03.50 Printed and Pubhshed m Ireland

© 1986 Elsevwr Scientific Pubhshers Ireland Ltd

age, whereas non-calcium-dependent release Is unaltered [1,2] The decline in calclun,dependent acetylchohne release is proportional to diminished calcium uptake by isolated nerve terminals [3] and matochondna [4,5] Calcium uptake by cultured skin flbroblasts also declines with the age of the donor [6] The reduced post-tetamc potenUatlon and the prolonged after-hyperpolarlzatlon in hlppocampal shces that accompany aging may be due to ~hanges in calcium homeostasis [7] Altered calcium metabohsm has also been Implicated in dementia [8] and age-related disorders, such as Alzhelmer's disease [6 9] The purpose of the present sludles was to determine if aging alters brain calcium metabolism m vtvo A prehmmary communication of some of these results was presented previously


Male CD-I mice (24 30 g) were from Charles River Laboratories lnc (Kingston, NY) Male BALB/cNNIA mice (3, 10 and 30 months of age) were from the National Institute on Aging Colony, which is contracted to Charles Rivers Breeding Laboratories (Wdmmgton, MA and Kingston, NY) The calcium-45 chloride (14 mC1/mg) and the hqmd scintillation cocktail, Aquasol-2, were from New England Nuclear (Boston, MA) The o-cresolphthalem complexone was from Sigma Chemical Co (St Louis, MO) The butterfly infusion sets (27 gauge, 4995-01) were from Abbott Hospital Products, Clucago, IL N-Tns(hydroxymethyt)methyl-2-ammo ethane sulfomc acid (TES) was from Sigma Chemical Co (St Louis, MO) The 1 5 ml Eppendorf polypropylene mlcrofuge tubes were from Walter Sarstedt (Princeton, N J) Methods

Sequential blood samples after rejection of *SCaC12 were obtained from a tad veto cannula, winch was made from a mo&fied butterfly infusion set The "wings" of the butterfly were cut and the extra tubing was removed Teflon tubing (28 gauge) was attached to the tubing, winch was connected to a stamless-steel y-connector with PE-50 tubing For isotope rejection, one part of the y-connector was attached to the syringe with the radioactive solution, while the other contained sahne to flush the cannula When blood was to be withdrawn, a 1-ml syringe was attached to the "flush" side of the cannula to apply a gentle vacuum About 15 ~1 of blood was collected m 1 5-ml mlcrofuge tubes After samphng, the cannula was flushed with hepanmzed (20 unlts/ml) sahne to prevent clotting m the hne During the samphng period, mice were either restrained in fenestrated cyhndncal holders or m a wire-mesh cage w~th their tads taped to the bench top Blood (10/A) and 0 4 N HC104 (10 tal) were added to mlcrofuge tubes, vortexed and then spun for 30 mln (12 000g) Ttus procedure breaks the blood cells and platelets and allows measurement of total blood calcium Radioactivity was determined in 10 tal of the supernatant by liquid scintillation Blood calcium was alsc~ determined m the acid supernatant by a spectrophotometnc assay, calcmm plus o-cresolphthalem complexone m an alkaline medium leads to a complex wtuch absorbs at 575 nm [11 ]

To remove any contribution of calcium or 4SCa from the blood to the brain value mice were perfused through a cannula m the aorta At the indicated tune interval, the mice were anesthetized with CO2, the chest cavity was opened and an 18-gauge needle was inserted and tied into the ascending aorta The right atrium was cut and the mouse was perfused for 3 mm (1 36 ml/min) with heparlnized (20 units/ml) saline with or without 100 mM CaC12 by means of a Harvard pump The presence of CaC12 m the perfusion solution did not appear to alter the amount of 4Scalcium that remained in the brain, the values [dpm/mg protein (n - 3), with or without calcium m the perfusion media respectively] were similar m whole cortex (1037 -+ 46 or 1238 + 21), or in 3 subcellular fractions P1 (1215+109 or 1351+72), P2 ( 1 1 9 9 + 4 4 or 1 3 3 7 -+13) or $2 (1138 + 135 or 1468 + 274) Since these results demonstrated that calcium was not removed from the brain in the absence of calcium in the perfusion solution, calcium was not added to the perfusion media in subsequent experiments Following perfuslon, brains were dissected into seven brain regions, as described previously [12] cortex, hippocampus, stnatum, cerebellum, brainstem, midbrain and forebrain If only radioactivity was to be determined, the tissue was weighed and solubihzed in l N NaOH or protosol and the d p m were determined by hquld scintillation with quench correction by external standardization The cortices were fractionated by standard methods [3] Tissue was homogenized in 0 32 M sucrose, 5 mM TES (pH 7 4) and centrifuged (3000 g for 3 rain) The pellet (P1, myelin, nuclei and tissue debris) was solubihzed in 2% deoxycholate in 1 N NaOH and ahquots were taken for determination of radioactivity and protein The supernatant was centrifuged at 17 000 g for 10 min The pellet (P2, synaptosomes, mltochondria and myehn) and supernatant ($2, microsomes, ribosomes and the cytosohc fraction) were solubihzed with 2% deoxycholate in 1 N NaOH for protein and radioactwlty determinations For measurement of brain calcium content, cortex was homogenized in 2 m! of 0 2 N trlchloroacetic acid, whereas all other regions were homogenmed in 1 ml Each region was homogenized (50 strokes) with a mechanical homogenizer and then spun at 17 000 g for 10 rain Calcium in the supernatant was determined by atomic absorption spectrophotometry (Perkin Elmer model 360) by comparison to CaCOa standards m 0 2 N trichloroacetic acid The protein m the pellet was deterrmned by the biuret method [13] after solublhzation in 1 N NaOH with 2% deoxycholate All statistlcal comparisons were by analysis of variance with the least sigmficant difference test RESULTS Assessment of calcium uptake into the brain requires knowledge of the blood specific actmty, as well as dpm m the brain If clearance of blood ~SCa is reduced and calcium specific a c t m t y stays elevated for longer tunes with a partlcular variable such as aging, more 4SCa is available to the brain for longer times so that absolute brain dpm without correction for blood specific actmtles does not accurately assess brain uptake Thus,






2'~ "o 2
















TIME (hr)


( each value is the mean of 5 mice )


Fig 1 Blood calcium disappearance curve CD-1 mice were rejected intravenously at 0 time w i t h 25 ~C1 o f 4SCa and blood was collected at the indicated times (A) Shows the results with a single mouse (B) Values means _+ S E M o f 5 mice In both A and B, blood samples were obtained at 1 2, 4, 6, 8 10, 15, 30, 45, 60, 120, 180, 240 and 300 m m


m io


3 o





TABLE I RATE OF BRAIN Ca.LCIUM UPTAKE WITH TWO DOSAGES OF 4SCALCIUM 25 vCt/mouse Blood spectfic activity at 5 h [(dpm/t~mote calcium) (10-s)] lntegralofbloodspectficacttvttyafter5h [(dpm/mg Ca) (mln) (10-1°)] Bratn 4SCa concentration [dpm/mg protein] Cortex Strlatum Brain 4SCa uptake

Cortex Strlatum

5 1 _+ 1 1 876_+ 042

1461 1083

_+140 _+ 68

50 vCt/mouse

12 2 _+ 1 0 13 17_+ 03

3339 2598

_+ 79 _+132

[(bram dpm/mg protem) (10~)] Integral of blood speclhc act|vlty 167_+ 0 16 1 2 4 _+ 010

254_+ 197_+

003 010

276_+ 210_+

021 016

[(bram dpm/mg protem) (103)[ [blood specific acnvlty at 5 h I Cortex Str~atum

265_+ 223_+

018 031

CD-I mice (n = 4) were rejected intravenously with the indicated ,o,CI of 4SCaCl2 at 0 time Sequential blood samples were taken as m big lb and ammals were sacrlfaced alter 5 h I lye-hour blood samples were obtained from the heart before perfusmn with sahne The integral ot the blood disappearance curve was determined graphically

estimation of uptake requires the brain dpm to be dehvered by an approximation of the integral o f blood specific activity A procedure was developed to allow the calculation o f brain calcium uptake m individual mice Since sequential samphng problems in aged mice prevented aqulsltlon of a complete time course of blood calcium specific activity, a method that utilized samples obtained at 5 h was compared to one In which numerous times intervals were monitored A blood disappearance curve could be obtained from a single young adult CD-1 mouse (Fig 1A) with highly rephcable results between mice (Fig 1B) Calcmm uptake into the brain increased over a 5-h period (Fig 2) If the amount o f isotope that was injected was increased twofold, the dpm In the brain and the blood at 5 h after Injection essentially doubled (Table I) Uptake was converted to approximate rates by dividing the d p m m the brain by the integral o f the blood specific actlwty or the blood specific actwlty at 5 h These m m a l studies estabhshed that blood specific activity at 5 h could be used to correct the rate o f calcmm uptake ff a varied amount o f isotope were injected (Table I) Thus, ff the dpm/mg protein were divided by the integral of the blood specific activity or blood specific activity at 5 h, the estimated rates were nearly independent o f the 4SCa








loo > I-. 0


E3 8

[] "O

0 UJ

[] o °

Q 4-

[] []



o 4o ,~ o z



TIME (hours)

I lg 2 The relation ot blood and brain calcium homeostasis m CD-1 mice 4SCa (25 ~tC1)was rejected intravenously at 0 time and individual mice were sacrltlced at the indicated times Blood was taken from the heart and the brain was then perfused with sahne before removal The brain was solubfllzed with 1 N NaOH, and the accumulated d p m were determined by hquld scintillation

dosage The correction with the 5-h sample gave a more consistent estimate than the use of the integral o f the blood disappearance curve Tins may have been because the early txme points, winch represent a large fraction of the area under the curve, affect the final result considerably and have the greatest vanatxon F o r example, the mean value for the 2 mm time point w~th the 50/aCl mouse dosage [(67 + 24)(107)dpm/mg calcium] and 25 /~C1 dosage [(18 +- 6)(107) dpm/mg calcium] had larger variations than the 5-h time points that are presented m Table I Once basehne conditions were estabhshed, 3-, 10- and 30-month-old BALB/cNNIA mice were injected esther intravenously or mtrapentoneally with 45CAC12and both modes of rejections revealed the same effect of aging Sequential samphng problems m aged mxce prevented aqulsltlon o f a complete time course, so that all results related to agang were calculated by dividing the dpm m brain by the blood calclum specific actlxaty at 5 h Estimated calcium uptake by various brain regions dechned with age whether the isotope was administered mtrapentoneaUy (Table II) or intravenously (Table I I I ) e v e n




StHatum Hlppocampus Cortex Cerebellum Forebram Bralnstem

3 months

10 months

30 months

154_+021 1 70+024 156_+019 1 80+024 192+027 1 97-+0 14

104+008 1 24_+013 104_+010 1 37_+011 1 3 5 + 0 12 160-+008

075 085 088 089 094 1 09

_+ _+ _+ _+ _+ -+

021 a 022 a 023 a 025 a 022 a 024 a

a Denotes dfllerence (P < 0 05) trom value for 3-month-old mite *SCalcmm (25/aCt/20 g mouse) was rejected mtrapentoneally and mice were killed 5 h later Values (means + S E M of 4 BALB/cNNIA mice) are bram dpm


mg protem

tzmole blood Ca

× 102

blood dpm

t h o u g h absolute u p t a k e values d i f f e r e d

B e t w e e n 3 and 10 m o n t h s , u p t a k e d e c h n e d

a p p r o x i m a t e l y 30% and a f u r t h e r 30% d e c h n e o c c u r r e d m 3 0 - m o n t h - o l d mice A f t e r an m t r a p e n t o n e a l r e j e c t i o n , t h e decrease was similar m all regions, w h e r e a s the f o r e b r a m TABLE Ili IN VIVO CALCIUM UPTAKE WITH AGING AFTER AN INTRAVENOUS INJECTION OF 4SCALCIUM



Stnatum Hippocampus Cortex Cerebellum Forebram Mldbraln Bramstem

3 months

10 months

30 months

2 25 + 0 2 75 +0 4 47_+0 2 58 + 0 2 97 + 0 2 49 + 0 1 32_+0

1 55 _+0 11 a 1 95_+0 16a 2 35 _+0 06 a 1 98 _+0 26 2 10_+0 16a 1 40 + 0 35 a 0 85 _+0 15a

1 17 1 51 3 86 1 30 1 20 0 82 0 51

06 17 48 25 09 38 14

4SCalcmm (25 ~C1/20 g mouse) was rejected intravenously and mice were killed 5 h later Values [means +- S E M of 4 (10 months) or 5 (3 and 30 months) BALB/cNNIA mice] are brain dpm mg protein

/amole blood Ca ×

blood dpm

X 10 a

a Denotes a difference (P < 0 05) from the value for 3-month-old mice b Denotes a difference (P < 0 05) from the value for 3- and 10-month-old ammals

-+0 05 a'b _+0 11 a _+0 76 _+0 14 a -+0 29 a'b _40 21 a + 0 06 a




P~ P2 S2

3 months

10 months

30 months

3 95 _+017 394_+017 163+_014 (5/

2 96 +-0 35 a 280+-020 a 1 08+-012 a ~4)

198 ±016 a 209±017 a 075±008 a (5)

Mice (BALB/cNNIA) were rejected Intravenously with 25 taC1 ol *S(a at 0 time and sacrificed 5 h later Alter pertuslon P1, P2 and S 2 were Isolated as described m the Methods section a Denotes difference (P < 0 05) from 3-month value Values (mean _+S E M of the n within parentheses) are brain dpm


mg protein

jumole blood Ca

x 103

blood dpm

( - - 6 0 % ) , m ~ d b r a m ( - - 6 7 % ) a n d b r a l n s t e m ( - - 6 1 % ) d e c h n e d (% decrease b e t w e e n 3 a n d 3 0 m o n t h s ) m o r e t h a n t h e o t h e r regions a f t e r a n i n t r a v e n o u s m F c t l o n The age-related d e c h n e m u p t a k e i n t o e a c h o f t h e subcellular f r a c t i o n s f r o m c o r t e x was similar

I n c o r p o r a t i o n o f c a l c m m i n t o P1 a n d P2 was q m t e slmllar a n d was m u c h

greater t h a n t h a t i n t o $2 T h u s , at 3, 10 a n d 3 0 m o n t h s i n c o r p o r a t i o n ( r e s p e c t w e % o f 3 - m o n t h - o l d m i c e ) decreased i n t o P1 ( 1 0 0 . 75, 50%). P2 ( 1 0 0 , 71, 53%) a n d $2 ( 1 0 0 . 66 and 46%) (Table IV)


Blood Cortex Strlatum Hlppocampus Cerebellum Forebram Mldbram Bramstem

3 months

10 months

30 months

1 0_+01 13 8 _+ 1 4 67+10 75 +10 46 +03 71_+05 133+18 96+1 7

10±01 13 5 +_06 77_+1 2 94±06 79±1 8 86±09 88±1 8 100_+14

09+_01 12 6 ± 1 0 49+06 70±08 54±06 74±07 50±10 a 5 0 ± 0 4 a'b

Values (~mole/ml ot blood, nmole/mg of protein) are means +- S E M ot 4 - 5 samples of blood and 6 for the brain from BALB/cNNIA mice a Denotes difference (P < 0 05) lrom 3-month value b Denotes difference (P < 0 05) from 3- and 10-month value

This &mmished calcium uptake by the brain dad not appear to be related to a dechne m the calcmm concentrations m the blood, although calcmm content in some brain regions dlrmmshed with age (Table V) Calcmm concentrations in blood were nearly identical at 3, 10 and 30 months of age Although calcium decreased slgmficantly by 30 months m bramstem (--62%) and midbram (--48%), the dechne in strlatum (--27%) was not significant and reductions did not occur in the other regions Altered calcium homeostasis with aging was also apparent from blood measurements For example, the specific activities [(dpm//amole Ca + S E M ) (×106)] at 5 h after an intravenous rejection increased (n = 4 - 5 ) with age from 0 63-+ 0 04 (3 months) to 1 34-+ 0 09 (10 months) to 1 86 +- 0 09 (30 months) Thus, the calcmm disappeared from the blood more slowly in senescent animals Therefore, an accurate estimate of brain uptake requires that blood specific activity be taken into account DISCUSSION The current results demonstrate that, within the hmltatlons of this method, m v t v o calcmm uptake into various brain regions dechnes during aging This reduction parallels the &mlmshed calcium uptake that occurs m v t t r o by synaptosomes [3] or into mltochondna [4,5] For example, the dechne m whole brain calcmm uptake m v i t r o was 55% between 3 and 30 months [3] and the decrease m the zn v t v o uptake m the current stu&es between those same ages was about 50% The m v t v o dechne in calcmm uptake also parallels the age-related decrease m acetylchohne synthesis m v l v o [14] Acetylchohne synthesis dechned about 40% and 60% at 10 and 30 months, respectively, and the correspondmg decreases of t n v t v o calcmm uptake were 30% and 50% The dechne m uptake Is also correlated to diminished tightrope test [14] and 8-arm maze [15] performance The age-related dechne m both of these behaviors can be partially amehorated by 3,4-dlammopyrldme, which promotes nerve terminal calcmm mflux and acetylchohne release [3,16] A high magnesmm dlet, which alters calcmm dynamics, can Improve age related deficits m calcmm-dependent electrophyslological events and memory [17] Together, these results strongly support the hypothesis that age-related alterations m calcium homeostasis are physiologically important The relation of calcium uptake by the brain to various serum compartments is complex [18] Serum calcium exists m a protein-bound non-&ffusable fraction (approx 40% of the total), a dlffusable lomzed free calcium (approx half of the total) and calcium complexed to anions such as bicarbonate and citrate The age-related dechne m serum calcmm m humans [19,20] is attributable to a decline m protein-bound calcmm as well as lomc calcium [19] The lomzed calcium Is generally regarded as the important physiological fraction, whereas the protein-bound fraction is thought to be more inert The measurement of these various pools is complex and uncertam [18] The interaction of these pools has not been well documented For example, calcium specific activity m urine and serum Is the same m normal animals, but much &fferent m starved animals which implies compartmentatlon of calcium in the serum [21] I n v t t r o mvestlgations

10 make It seem unhkely that the protein-bound and dlalysable calcium contribute to urine but suggest that a complexed form such as calcium citrate may [21] After Injection of 4SCa, certain tlssues (e g skin, lung and heart) show specific activities tugher than In circulating blood at earlier times, whereas others (e g brain and muscle) only equilibrate very slowly [22] If, in the present studies, the rejected 4SCa mainly equlhbrated with the ionized pool In the blood within the experimental times utilized, then the apparent effects of aging on calcmm uptake Into the brain would be even more than the present calculations suggest (1 e the specific activity of plasma calcmm would have been much lugher) Further experiments are required to determine how aging affects the partlonmg of 4SCa between these various compartments in blood Calcmm homeostasis m the brain is also influenced by the uptake across the arachnold membranes and choroId plexus into the cerebrosplnal fluid (CSF). as well as across the cerebrovascular endotheha [23] The CSF maintains brain calcium levels despite fluctuations m serum levels Approximately 50% of brain calcium originates In the CSF [24], so that tlus may make considerable contribution to brain uptake If CSF specific activity IS similar to serum specific activity, the conclusions of the current study with regard to the effects of aging would be correct Further experiments would have to determine if calcium transport into the CSF and across the cerebrovascular endothehum was also altered by aging Calcium uptake by brain m w v o has been examined wlth agtng m one previous study I n v t v o 4SCa uptake by rat brain, at 48 h after Injection, was similar or increased between 4 and 24 months, even though brain calcmm content increased [25] However, these results were not corrected for the disappearance of 4SCa from the blood and the time between ISOtope injection and sacrifice exceeded the hnear uptake period Since the disappearance curves and 5 h time points were crmcal in dehneating the decreased uptake In the current studies, it is not clear If these two studies are contradictory Furthermore~ the animals In the previous studies were not perfused If the blood 4SCa remained elevated, as In the current studies, this may have resulted In an apparent age-related increase The higher blood 4SCa specific activity In the aged ammals m the current study suggests that the clearence of 4SCa2+ by the kidney, which also requires non-protein bound calcmm, IS slowed with age Thus, any study on tissue uptake requires that blood specific activity be taken into account The effect of aging on m w v o brain calcmm content IS surprisingly controversial The frequency of spontaneous mmerahzatIon of the brain Increases with age [26] In elderly humans, a mild degree of calcification occurs In and around blood vessels of the basal gangha, Including the thalamus [27] X-Ray mIcroanalysls detects deposits with variable amounts of calcium on the surface of vascular basement membranes [28] Examination of the minerahzed deposits indicates that calcium binds first to siahc acid and later to phosphate groups [29] Increased brain calcium content has been reported In dogs, guinea pigs [30], humans [31,32] and rats [25,33], although many of the early studies had large variations m the measurements Other results found no difference m rat brain calcium content with aging [34,35] The excess calcium that some report may be mainly

11 extraceUular [28,35]

The present studies demonstrate that when the brain is perfused

either a dechne or no change in regional calcium content accompames aging in mice Altered calcium homeostasis has been imphcated in several age-related impairments, but whether it is a cause or an effect remains to be estabhshed Deficits m calcium uptake m vtvo may be because of or lead to decreased calmoduhn [36] and the diminished

calcium uptake m vitro [3,37]

A similar difficulty with cause-effect relations apphes to

the reduced potentiatmn and prolonged after-hyperpolarization in luppocampal shces that accompames aging [7]

Although hypocalcemm is associated with dementia [8]

further studies are required to estabhsh any cause-effect relations AC KNOWLEDGE MENT S The authors gratefuUy acknowledge Dr E Windhager and Dr G Frindt for the use and training with the atomic absorption spectrophotometer Supported in part by grant NS03346, AGO4177 and the Winifred Masterson Burke Rehef Foundation

REFERENCES 1 G E Gibson and C Peterson, Aging decreases oxidative metabohsm and the release of acetylcholine J Neurochem , 37 (1981) 978-984 2 F Pedata, J Slavlkova, A Kotas and G Pepeu, Acetylchohne release from rat cortical shces during postnatal development and aging Neuroblol Aging, 4 (1983) 31-35 3 C Peterson and G E Gibson, Aging and 3,4-dlammopyndme alter synaptosomal caEmm uptake J Btol Chem, 258 (1983) 11482-11486 4 S W Leslie, L J Chandler, E Barr and R R I,arrar, Reduced caloum uptake by rat brain mltochondrta and synaptosomes m response to aging Beam Res, 329 (1985) 177-183 5 C Peterson, D G Nleholls and G E Gibson, Subsynaptosomal distribution of calcmm during aging and 3,4-dlammopyndme treatment Neuroblol Agmg, 6 (1985) 297-304 6 C Peterson, G E Gibson and J B Blass, Altered calcmm uptake m cultured skin fibroblasts from patients with Alzhelmer's disease N Engl J Med, 312 (1985) 1063-1064 7 P W Landfield and T A Pitier, Prolonged Ca~+-dependent after hyperpolanzatlons an hlppocampal neuron of aged rats Scwnce, 226 (1984) 1089-1091 8 P G Ettlgl and G M Brown, Brain disorders assocmted with endocrine dysfunction Psychzatr Chn North A m , 1 (1978) 117-136 9 D P Perl, D C Gajdusek, R M Garruto, R T Yanagthara and C J Gibbs, Aluminum accumulation m amyotrop2c lateral sclerosis and Parkmsomsm dementm of Guam Scwnce, 21 7 (1982) 10531055 10 G Gibson, P Perrmo and G Dlenel, Alterattons of m vwo brain calcmm homeostasis with aging Age, 14 (1984) 62 (Abst) 11 B Sarkar and U Chauham, A new method for determining micro quantmes of ~alctum m biological materials Anal Btochem, 20 (1967) 155 - 166 12 C Peterson and G E Gibson, 3,4-Dmmmopyrtdme alters acetylchohne metabohsm and behavior during hypoxla J Pharmaeol Exp Ther , 222 (1982) 576-582 13 A G Gomall, C J BardawtU and M M David, Determination of serum proteins by means of the bmret reaction J Blol Chem , 1 77 (1949) 751-766 14 G E Gibson, C Peterson and D J Jenden, Brain acetylchohne synthesis dechnes with senescence Scwnce, 213 (1981) 674-676 15 H P Davis, A ldowa and G E Gibson, Improvement of 8-arm maze performance m aged 1 lsher 344 rats with 3,4-dlammopyndme Exp Aging Res, 9 (1983) 211-214

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