Neurobiology of Aging, Vol. 8, pp. 147-152. ©PergamonJournals Ltd., 1987. Printedin the U.S.A.
0197-4580/87$3.00 + .00
Selective Alteration of Mouse Brain Neurotransmitter Release With Age G A R Y B. F R E E M A N A N D G A R Y E. G I B S O N 1
Cornell U n i v e r s i t y M e d i c a l College, B u r k e R e h a b i l i t a t i o n C e n t e r 785 M a m a r o n e c k A v e . , W h i t e P l a i n s , N Y 10605 R e c e i v e d 9 April 1986 FREEMAN, G. B. AND G. E. GIBSON. Selective alteration of mouse brain neurotransmitter release with age. NEUROBIOL AGING 8(2) 147-152, 1987.--The release of acetylcholine (ACh), glutamate (GLU) and dopamine (DA) from various brain regions was investigated in young (3 month) and old (30 month) Balb/c mice. Aging increased the basal release of GLU (77%) and DA (29%) in striatum and GLU in hippocampus (94%); the concentrations of these neurotransmitters in the media after K + stimulation were unaltered by aging. Although the basal release of ACh was not altered by age, K+-stimulated ACh release was reduced in striatum. The age-related increases in basal GLU and DA release may be important in the pathophysiology of cell death during aging. Release
SELECTIVE alteration of neurotransmitter function may underlie age-related deficits in psychomotor performance  and cognition . Diminished in vivo acetylcholine (ACh) synthesis during aging without a reduction in levels suggests altered release . ACh release in vitro decreases with age after potassium stimulation in mouse whole brain  or rat striatal  slices and following K + or electrical stimulation of rat cortical slices [38, 41, 42]. Although turnover rates of dopamine (DA) decline in aged rats  and mice [14,15] both decreased [1, 3, 5, 50] and unchanged [31,44] levels have been reported. Although calcium-dependent, KCl-stimulated release of [3H]DA from 12 month old mice increases compared to 2 month old mice in forebrain synaptosomes , no impairment of release is apparent in striatum . Studies on alterations of glutamate (GLU) metabolism with aging are also inconclusive. Decreased GLU levels have been reported in aged rats [10, 11, 27, 54], but not in aged mice [23,32]. Synthesis of GLU in vivo from radiolabeled glucose either decreases  or remains unchanged [11,23] with age. In vitro GLU synthesis in cortical brain slices  and oxidation by rat brain mitochondria decreases during aging [6,12]. Low affinity uptake of GLU into rat cerebral cortical slices  and brain mitochondria  are markedly reduced, while high affinity uptake by striatal slices increases with age . Because of the apparent conflicts on the effects of age on various neurotransmitters in previous reports, the release of GLU, ACh and DA in the aged mouse was examined with a single release system. This was done in the striatum, hippocampus and cortex. The striatum is a particularly attractive area of the brain in which to study age-related changes because it contains high concentrations of ACh, DA and G L U and has repeatedly been shown to be susceptible to
various metabolic disorders such as hypoxia  and ischemia [ 16]. The striatum contains small intrinsic cholinergic neurons whereas the hippocampus and cortex have primarily nerve endings of cholinergic projections from other areas. The density of G L U receptor sites are highest in the hippocampus and substantial distribution is also observed in striatum and cortex . A sensitive in vitro release model, which helped to characterize the selective alteration of neurotransmitter release during anoxia , was used in the present investigation. This release system has a preincubation that is followed by K + depolarization without rinsing the tissue. The advantage of this system is that it allowed release to be studied with minimal handling of the tissue and provided maximal dopamine release and recovery. The disadvantage is that any neurotransmitter released during the preincubation is present during K+-stimulated release and can modify the subsequent release and treatment effects. These have been carefully documented elsewhere [17,18]. METHOD
Materials Male Balb/c mice (3 and 28-30 months) were purchased from the National Institute of Aging Colony which is under contract to Charles River Breeding Laboratories (Wilmington, MA). In the presentation of the results, the 28--30 month old mice are referred to as 30 month mice. [3Hacetyl]acetylcholine iodide (50-100 mCi/mmol) and Aquasol 2 were from New England Nuclear (Boston, MA). D[U-~4C]glucose (250-360 mCi/mmol) was from ICN Radiochemicals (Irvine, CA). Paraoxon was from K + K Laboratories (Plainview, NY). Prosil-28, an organosilane concen-
iRequests for reprints should be addressed to Dr. Gary Gibson.
FREEMAN AND GIBSON STRIATUM
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FIG. 1. Effects of age and K '-depolarization on regional brain GLU release. Clear bars represent basal release. Hatched bars are total media neurotransmitter after K + stimulation. Solid bars denote the groups that were stopped before the release incubation. Data was subject to age x K + stimulation analysis of variance. Striatal GLU release varied with age, F(1,28)-4.54, p<0.05, and K' stimulation. F(1,28)= 14.64, p<0.001. An age x K ~ stimulation interaction was not significant. Cortical release varied as a function of age, F(1,42)=9.31, p<0.005, and K', F(1,42)=19.33, p<0.001. Values (nmol per mg protein) are means+SEM of 7 8 mice per group, p<0.05. "Denotes a significant effect of K stimulation. "Denotes that the 30 month value differs significantly from 3 month. "Denotes that the value at end of basal release incubation differs from that at the end of the preincubation.
trate surface-treating agent was from Specialty Chemicals (Gainesville, FL). The electrochemical d e t e c t o r (LC-4B) and flow cell (LC-17) were from Bioanalytical Systems, Inc. West Lafayette, IN). The chromatographic column (RadialPak t~Bondapak C18 c a r t r i d g e , 8 mm), Z-module radial compression separation system, sample delivery system (model 710 B W I S P and 6000 A pump) and data reduction system (model 730 data module and model 721 system controller) were from Millipore, Waters C h r o m a t o g r a p h y Division (Milford, MA).
Tissue Preparation Each animal was decapitated and the striatum, hipp o c a m p u s and cortex were r e m o v e d bilaterally and placed into separate vials containing ice cold buffer (pH 7.4): 141.0 m M - N a C l , 5.0 mM-KC1, 2.3 mM-CaCl2, 1.3 mM-MgSO.~ and 10.0 mM-Na2HPO4 . The individual brain regions were sliced in two dimensions (0.3 mm intervals) with a McIlwain tissue chopper (Brinkman Instruments Inc., Westbury, NY), gently dispersed and then washed with ice cold buffer until the rinses were clear. Tissue slices were placed in siliconized scintillation vials in a final v o l u m e of 1 ml of incubation buffer. In addition to the c o m p o n e n t s listed above, the incubation buffer also contained 5 mM-D-[U-14C]glucose (1 /zCi/tzmole), 50/xM-choline chloride and 0.1 mM-cysteine.
Release System The vials with slices were sealed with serum stoppers with suspended Kontes cups that contained 0.2 ml of 1 M-hyamine hydroxide to trap 14CO., on fluted filter paper and flushed with 100% Oz for 10 min. After a one hour preincu-
bation at 37°C, the vials were quickly removed from the incubator, placed on ice and flushed with 100%, O~ for an additional 10 rain. Paraoxon (an acetylcholinesterase inhibitor; final c o n c e n t r a t i o n = 4 0 p~M) and either KCI (10 /~1=26 /xmoles) or distilled water (10/~l) were added simultaneously at the end of the second aeration. The vials were then incubated for 5 rain at 37°C. At the end of the release incubation, [:~H]ACh (approximately 7000 dpm) was added and 970/.d of the contents of the vial were transferred to a 1.5 ml microfuge tube containing 400 pJ of a silicone oil:dinonylphthalate solution (50:50). The tubes were spun (15,000 z g, 30 sec) in an E p p e n d o r f microfuge and the neurotransmitters in the supernatant above the oil were analyzed. The quantity of tissue slices for incubations was regiondependent. Cortical yield was adequate to permit a normal and high K* group as well as samples that were stopped before the release incubation. H o w e v e r , the yield of tissue slices from a single striatum or hippocampus allowed only normal and high K + release incubation groups to be run. In experiments in which the release methods for DA, ACh and G L U were standardized for striatal slices, low K ~ and high K* release groups as well as samples that were stopped prior to the release incubation (i.e., after the aeration on ice) were directly compared. For each neurotransmitter in the striaturn, levels in the media after a 60 min preincubation were similar to low K ~ samples that were incubated for an additional 5 min [DA(pmol/mg protein): 7.07_+1.08 versus 5.45+-0.81; ACh(dpm/mg protein): 658_+46 versus 633_+51; G L U ( n m o l / m g protein): 1.44+-0.10 versus 1.28+_0.09; for low K ~ samples stopped before or after the release incubation, respectively, n - 1 2 per group]. The samples from within each age group were not pooled so that an indication of the
RELEASE AND AGING
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3 30 AGE (months)
FIG. 2. Effect of age and K + on regional brain ACh release. Clear bars represent basal release while hatched bars are total media neurotransmitter after K ÷ stimulation. Data was subject to age × K + stimulation analyses of variance for striatum and cortex and a one-way analysis of variance for hippocampus. A significant K + effect, F(1,28)=68.56, p<0.001, and age x K + interaction, F(1,28)=4.30, p<0.05, were indicated in the striatum. Values (dpm per mg protein) are means_+SEM of 7-8 mice per group, p<0.05, aDenotes a significant effect of K + stimulation. "Denotes that the 30 month value differs significantly from 3 month.
variability from animal to animals within each group could be determined.
ACh, DA, GLU and Protein Determinations An aliquot (345/zl) of the supernatant a b o v e the oil was acidified by the addition o f 23 p,l o f 0.4 N-perchloric acid (final c o n c e n t r a t i o n = 0 . 0 2 5 N - P C A ) and 120/zl was directly injected on to an H P L C for determination of D A by liquid c h r o m a t o g r a p h y with e l e c t r o c h e m i c a l detection . The remainder of the aliquot (200 gl) was used to determine G L U by e n z y m a t i c assay . The rest of the supernatant (500/zl) was r e m o v e d and c o m b i n e d in a conical glass centrifuge tube with 1.5 ml of ice-cold rinsing buffer and [3H]- and [14C]ACh were extracted . The dpm were determined in a B e c k m a n LS 9000 liquid scintillation system with correction by external standardization. The tips of the microfuge tubes containing the slices were cut and placed into separate tubes containing I ml of 2% d e o x y c h o l a t e in 1 N - N a O H . The tubes were heated at 60°C until the tissue dissolved. The protein content was determined by the biuret reaction with bovine serum albumin as the standard .
Statistical Analysis Data was analyzed with separate age x K + stimulation analyses o f variance for each brain region. Statistical comparisons a m o n g specified m e a n s were done using multiple t-tests . RESULTS Selective presynaptic changes in G L U release with age o c c u r r e d in the three brain regions (Fig. 1). M e d i a G L U concentrations were l o w e r in c o r t e x than in striatum or hippocampus. Although total G L U under high K + conditions did not vary with age in any of the regions, 30 month animals had increased basal (normal or low K ÷) release in striatum
AGE (months) FIG. 3. Effect of aging and K ÷ depolarization on dopamine release in striatum. Clear and hatched bars represent basal and total media dopamine after K + stimulation, respectively. An age × K + stimulation analysis of variance indicated a significant effect of only K + stimulation, F(1,28)=74.89, p<0.001. Values (pmol per nag protein) are means_+SEM of 7-8 mice per group, aDenotes a significant K + effect, p <0.05. hStatistical comparison by a separate t-test indicated a significant increase of basal (low K +) DA release in 30 month mice, t(12)=2.21, p<0.05.
F R E E M A N A N D GIBSON
and hippocampus (p<0.05). Thus, the percent stimulation of G L U release by K ÷ decreased (/9<0.05) with age in striatum (3 month, 111_+26%; 30 month, 38_+21%). Cortical G L U concentrations in the group that was stopped before the release incubation increased with age, which suggests increased release during the preincubation. However, by the end of the release incubation, no age-related difference was apparent in media G L U during basal or K+-stimulated conditions. Thus, the difference between normal K ÷ samples and those stopped before the release incubation was significant in cortex of 30 month mice (p<0.05). ACh release also changed with age (Fig. 2). In striatum, basal ACh release was similar in 3 and 30 month mice whereas total ACh under high K ÷ declined in the 30 month group (p<0.05). Cortical ACh release increased similarly after K + stimulation in both age groups. Reliable counts (dpm) for hippocampus were obtained for the high K + condition only; aging did not effect hippocampal K+-stimulated ACh release. Changes in DA release also occurred with age (Fig. 3). Measurable quantities of DA were present in striatum after basal and K+-stimulated release whereas cortical values were only detectable after K + stimulation and hippocampal release was not detectable. An increase (29%) in basal DA release in the striatum was observed in 30 month old mice. K + depolarization increased striatal DA release in both young and aged animals. High K + values for cortex (pmol per mg protein; n = 7 - 8 per group) were 9.4_+2.1 (3 month) and 13.8-+3.1 (30 month). DISCUSSION Age-related differences in the regulation of neurotransmitter release were region and transmitter specific. Thus, basal G L U release increased in striatum and hippocampus while levels after K+-stimulation were unaffected. K +stimulated ACh release declined in striatum, but not in cortex or hippocampns. Basal DA release increased in striatum whereas K+-stimulated DA release did not differ significantly with age in any region. The lack of a significant decrement in striatal K+-stimulated DA release agreed with earlier findings . The absence of a K+-stimulated deficit in ACh release in cortex with age was at variance with previous findings in rats [38, 41, 42]. However, striatal K +stimulated ACh release was sensitive to age and suggests the possibility of species differences in regional cholinergic deficits with age. The effects of altered release of neurotransmitters on their extracellular concentrations depends upon reuptake processes. Although reuptake processes may be involved, in young CD-1 mice during the final 5 min incubation, DA release exceeds reuptake by a ratio of 33:1 (low K +) and 50:1 during high K + . In contrast to DA, recent experiments in the striatum have shown smaller ratios of release to reuptake for G L U (1.8:1 in low K + samples and 2.3:1 in high K+). Therefore, reuptake processes may be more important in GLU metabolism and, thus, elevated striatal and hippocampal GLU in low K + media of 30 month old mice may be due to increased release or decreased reuptake. In vitro, low affinity uptake into cortical slices declines , while high affmity uptake by striatal slices  increases with age. The significantly higher G L U in the cortex of 30 month old mice at the end of the preincubation is consistent with decreased reuptake. However, G L U concentrations are subsequently reduced in the low K + release incubation to levels of 3 month mice which suggests that G L U reuptake may be increased in aged mice. R.euptake may
have been altered early during the preincubation and after 60 min any indication that changes in reuptake had occurred would not be apparent. Thus, increased release likely accounts for the high levels of G L U in the media of aged animals. The influence of non-release incubated blanks on the interpretation of the data demonstrates a critical need for the inclusion of this group in studies of release mechanisms by this method. In addition, shorter release incubations may provide a better indication of changes in reuptake processes. A prolonged elevation in extracellular G L U may play a role in the degenerative changes associated with normal aging. This may occur through an excitotoxin mechanism as has been proposed for G L U during hypoxia/ischemia . The nucleus basalis receives glutamatergic input . Stereotaxic injection of kainic acid or quinolinic acid, which stimulate G L U receptors, into the nucleus basalis of the rat causes neuronal destruction and a reduction in the projection area of these cells of several cortical cholinergic markers including choline acetyltransferase, acetylcholinesterase, choline uptake and [3H]ACh release [4, 13, 29, 33]. Increased basal release of G L U in the striatum and hippocampus along with a reduction of K+-stimulated ACh release in the striatum may be indicative of a similar series of events during normal aging. Intrastriatal injections of GLU, kainic acid or quinolinic acid lead to similar losses of cholinergic markers in the striatum [7, 35, 40, 47]. Although the present study was conducted with normal aged mice, alterations in G L U release may have important implications in the pathogenesis of age-related neurodegenerative disorders [7, 24, 35, 40]. Excessive DA release may lead to cell death in the same manner as excessive G L U release. The increase in basal DA release in 30 month mice suggests that prolonged stimulation by DA during aging may cause cell death or alter DA receptors. A loss of striatal D2 receptors that occurs during aging in the mouse [45, 48, 49] and in patients with Alzheimer's disease  may reflect such cell loss. The present report used a release system that measured media concentrations of neurotransmitters following incubation of tissue slices. Results showed that G L U and possibly DA homeostasis are altered with normal aging and suggest the involvement of these neurotransmitters in neurodegenerative disorders of aging. Since release was assessed by measuring neurotransmitters in the media, tissue integrity and other aspects of neurotransmitter metabolism may influence the interpretation of these results. These other alternatives include age-related changes in the pool of transmitter available for release, reuptake systems, feedback inhibition mechanisms and the sensitivity of autoreceptors regulating release. Regardless of the mechanism, aging appears to increase the extracellular concentration of G L U and DA. This may be important in the production of cell death through an excitotoxin mechanism . The decreased release of ACh, on the other hand, may be important in the age-related impairment of cognition.
ACKNOWLEDGEMENTS This work was supported in part by grants AG04171 and AG05352 from the National Institutes of Health and the Winifred Masterson Burke Relief Foundation. The authors thank Peter Perrino and Victoria Mykytyn for technical assistance.
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