- Email: [email protected]
Fischer 344 hybrid rats
Brain Research 791 Ž1998. 246–256
Research report
Age-related decline in striatal dopamine release and motoric function in Brown NorwayrFischer 344 hybrid rats David M. Yurek
a,b,)
, Susan B. Hipkens a , Meleik A. Hebert c , Don M. Gash b , Greg A. Gerhardt
c
a Department of Surgeryr Neurosurgery, UniÕersity of Kentucky College of Medicine, Lexington, KY 40536, USA Department of Anatomy and Neurobiology, UniÕersity of Kentucky College of Medicine, Lexington, KY 40536, USA Departments of Psychiatry and Pharmacology, Neuroscience Training Program and the Rocky Mountain Center for Sensor Technology, UniÕersity of Colorado Health Sciences Center, DenÕer, CO 80262, USA b
c
Accepted 27 January 1998
Abstract The Brown NorwayrFischer 344 F1 hybrid rats ŽF344BNF1 . is a newer rat model and is emerging as an important rodent model of aging. In the present study we used motoric performance tests, intracerebral microdialysis, and neurochemical measures of postmortem brain tissue to investigate the effects of aging in young Ž4–5 months., middle-aged Ž18–19., and old Ž24–25 months. F344BNF1 hybrid rats. We observed that old F344BNF1 rats exhibited decreased motoric performance, and lower levels of spontaneous and Damphetamine-induced locomotor activity than those observed in young F344BNF1 rats. Microdialysis measures of extracellular basal levels of dopamine ŽDA., 3,4-dihydroxyphenylacetic acid ŽDOPAC., and 4-hydroxy-3-methoxyphenylacetic acid ŽHVA. were significantly diminished in the striata of the middle-aged and old rats as compared to levels in young animals. In addition, D-amphetamine-evoked overflow of DA was significantly decreased in the middle-aged and aged rat striatum as compared to DA overflow in young F344BNF1 rats. Studies of postmortem brain tissue showed that the changes in overflow of DA correlated with significantly lower DA tissue content in ventral striatum and midbrain. Moreover, both dopamine turnover ratios ŽDOPACrDA, HVArDA. and the serotonin turnover ratio Ž5-HIAAr5-HT. were significantly elevated in the ventral striatum and nucleus accumbens. The results of this study demonstrate a correlation between reductions in striatal DA neurochemistry and diminished motor function in aged F344BNF1 rats. q 1998 Published by Elsevier Science B.V. Keywords: Aging; 3,4-Dihydroxyphenylacetic acid; 4-Hydroxy-3-methoxyphenylacetic acid; D-amphetamine; Microdialysis; F344BNF1 rat
1. Introduction Aging in humans is associated with a decline in general motor skills w3x. The factors that produce motor dysfunction in aging are thought to be neurological in origin, and appear to be associated with a decrease in nigrostriatal dopaminergic activity w3,21x. Morphological studies indicate that DA neurons in the human midbrain may be lost at a rate of 4–6% per decade from the ages 20 to 90 during the normal course of aging w9,26x. Dopamine ŽDA. content in the caudate nucleus is decreased in aged humans w6x. Kish et al. w21x reported a 60% decline in striatal DA levels in normal subjects within the age range of 14 to 82 years. ) Corresponding author. Dept. of SurgeryrNeurosurgery, University of Kentucky Health Sciences Research Building, Lexington, KY 40536-0305, USA. Fax.: q1-606-323-6343; E-mail: [email protected]
Additional markers of the DAergic system have been found to decrease over the same time period. Levels of tyrosine hydroxylase ŽTH. mRNA in the substantia nigra, the rate limiting enzyme in DA synthesis, appear to decline steadily from young adulthood to old age w2x. Also, reductions of DA high-affinity uptake sites have been observed in aged humans w1,44x. Levels of DA transporter mRNA in the substantia nigra are also markedly decreased with age w2x. Clearly, changes in DA neuronal systems correlate with reduced motor skills in aged humans, and may be responsible for age-related reductions in motor abilities of humans. Rodent studies over the last 2 decades have produced equivocal results concerning changes in the nigrostriatal system with age. Dopamine synthesis w43x, catecholamine levels and catecholamine turnover w32x, tyrosine hydroxylase activity w27,37x, DA and DA metabolism w8,11x, DA
0006-8993r98r$19.00 q 1998 Published by Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 1 1 0 - 3
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release w13,35,41x, and DA receptor binding w37x all show significant declines in the striatum of aged rats when compared to similar measures in adult animals. In contrast, other studies report no changes in measures of nigrostriatal systems during aging w5,13,13,19,28,29x. These differences may be related to the animal models and techniques used to study the aged brain. Most of the aforementioned studies that examined DA and DA metabolites levels in the striatum of aged rodents have measured the total intracellular and extracellular catecholamine content within tissue samples. At the present time, few studies have addressed the dynamic release and metabolism of DA in the striatum of aged rats w20x. The objective of the present study was to use intracerebral dialysis as a means to investigate basal and stimulusevoked overflow of extracellular levels of DA and DA metabolites in the striatum of young and aged F344BNF1 rats, and to characterize locomotor activity and tissue neurotransmitter levels in the same animals. We chose the F344BNF1 rat in these studies because it is a model of aging that may have some advantages over other strains currently used for aging studies. This strain of rat is preferred over other strains because of its normal distribution of age-related pathologies occurring relatively late in life w40x. The aged F344BNF1 also has a low incidence of renal pathology, a common problem in aged Fischer 344 rats w22x. In addition, this hybrid strain does not have specific tumor susceptibilities that occur in some of the other rat models of aging. Preliminary studies which have examined age-related changes in motor function have indicated that the F344BNF1 rat exhibits a greater reduction of locomotor activity with age than does the Brown Norway or Fischer 344 strains w39x. Moreover, few systematic functional and behavioral studies of the DA neuronal system have been carried out in this relatively new rat model of aging.
2. Methods 2.1. Animals Male F344BNF1 rats ŽNIA, Charles River. were used in this study. Young Ž4–5 months old, n s 20., middle-aged Ž18–19 months old, n s 16., and old Ž24–25 months of age, n s 19. rats were housed in 26 = 48 = 20 cm acrylic cages with wood chips for bedding, and food and water available ad libitum. Animal cages were kept in a vivarium room and shelved within an environmental hoodrlaminar flow facility where temperature Ž23 " 18C., humidity Ž53 " 15%., and lightrdark cycle Ž12 h light:12 h dark. were closely controlled. Tissue samples and rod walk data were obtained from one cohort of rats at each age group: young w n s 6x, middle-aged w n s 6x, or old w n s 6x. Locomotor activity and microdialysis were obtained from separate cohort of rats: young w n s 14x, middle-aged w n s 10x, and
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old w n s 13x. All animal use procedures were approved by the University of Kentucky Animal Care and Use Committee, and were in strict accordance with the NIH Guide for the Care and use of Laboratory Animals. 2.1.1. Rod walk test Motor coordination and balance were assessed using a rod walking test w42x. The testing apparatus consisted of a 60 cm long wooden dowel of 5 cm in diameter that was grooved and suspended between two platforms 60 cm above a padded surface. Rats were placed in the center of the rod facing one of the platforms. Each rat was allowed three attempts to reach the escape platform, with a maximum time limit of 120 s. An additional rank score was also determined using the following scale: 0—fall, 1— clasp, 2—all paws on top, 3—takes steps, 4—reaches platform. Rats were assigned the best score from the three individual trials w13x. Sample size for each age group: young w n s 6x, middle-aged w n s 6x, old w n s 6x. 2.1.2. Locomotor actiÕity testing Locomotor activity was tested in Digiscan Animal Activity Monitors ŽOmnitech Electronics.. Each monitor contains an array of infrared sensors that are arranged in vertical and horizontal planes and are capable of measuring several components of animal activity simultaneously. This study focused on four measures of motor activity: horizontal activity, total distance, movement time Žambulation time., average distance per movement. Horizontal activity is defined as the number of infrared light beam interruptions that occur on the horizontal sensor during the test period. Total distance is defined as the total number of centimeters traveled by the animal during the test period. Movement time is defined as the time, in seconds, that the animal is ambulating; if the animal stops ambulating for longer than 1 s then this parameter is not incremented until the next ambulatory movement is made. Average distance per movement is measured in inches and is calculated as the average distance each animal traveled per ambulatory movement; an ambulatory movement is considered terminated if the animal remains non-ambulatory for longer than 1 s. Digiscan Activity monitors were housed directly in the animal room. All activity measurements were made during the dark phase of the animal’s diurnal cycle. Spontaneous locomotor activity was monitored by placing the animal inside the activity chambers, habituating the animals for 15 min, and then monitoring activity for a 2 h period. The D-amphetamine-induced locomotor activity was monitored similarly with the exception that the animals were administered D-amphetamine Ž5.0 mgrkg, i.p.. immediately after the habituation period. Spontaneous and D-amphetamineinduced locomotor activity were assessed in the same animals with a 1 week period intervening between each test. Sample size for each age group: young w n s 14x, middle-aged w n s 10x, old w n s 13x.
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2.2. Intracerebral microdialysis Each animal was anesthetized with a halothane–air mixture Ž1.0–1.5% halothane at 1.5 lrmin., placed in a stereotax, and maintained under halothane anesthesia for the duration of the microdialysis sampling period. Body temperature was maintained at 378C using a heating pad ŽCMA 150 Temperature Controller, Carneige Medicin.. Concentric dialysis probes Ž3.0 mm membranes, 0.5 mm diameter, CMAr12, Carneige Medicine. were used for all intracerebral dialysis studies. Dialysis probes were stereotactically lowered into each striatum Žbilateral, q1.0 mm A–P, "2.5 mm ML w33x. until the tip of the dialysis probe was 6.5 mm below the dura. The inlet of the dialysis probes were connected to a microinjection pump ŽCMA 100, Carneige Medicine. and perfusate w147 mM NaCl, 1.0 mM CaCl 2 , 3.9 mM KCl, pH s 6.0x was continuously pumped into the probe at a flow rate of 2.0 m lrmin. The first sample period began 1.5 h after the probe was stereotactically lowered into the brain. Striatal perfusates were collected every 25 min in microcentrifuge tubes containing 10 m l of 0.1 M perchloric acid, as a preservative, and each sample was subsequently stored at y808C. Sample size for each age group: young w n s 14x, middle-aged w n s 9x, old w n s 13x. For measurements of D-amphetamine-stimulated overflow of DA, the flow of perfusate into the left dialysis probe was switched from perfusate only to perfusate containing 250 m M D-amphetamine sulfate for 25 min, and then switched back to perfusate without D-amphetamine; right dialysis probe was not switched to D-amphetamine. Samples were assayed for levels of DA, DOPAC, and HVA using HPLC with electrochemical detection described in Ref. w18x. Compounds were separated on a 150 = 3 mm MD-150 column ŽESA.. Mobile phase wpH s 3.0, 75 mM sodium phosphate, 0.1 mM EDTA, 3.0 mM octyl sodium sulfate, and 10% acetonitrilex was pumped at a rate of 0.6 mlrmin. The HPLC system was coupled to dual-coulometric detectors ŽModel 5014B, ESA. with a pre-conditioning electrode set at y175 mV and the detection electrode set at 150 mV. Dialysate values were corrected for probe recoveries at 378C and are reported relative to 100% recovery. Probe recoveries were in the range of 9–15% " 1.4% for DA and 15–21% " 1.9% for DOPAC and HVA. 2.2.1. Determination of monoamine and monoamine metabolite leÕels in brain tissues Following the behavioral studies, rats were decapitated and their brains were quickly removed and placed in ice-cold saline. After cooling, tissue samples of the medial prefrontal cortex, nucleus accumbens, dorsal striatum, ventral striatum, midbrain Žsubstantia nigra and ventral tegmental area combined., and the hippocampus from the young, middle-aged and old rats were dissected, placed
into storage tubes and weighed, and stored at y708C. Anatomical verification of microdialysis probe recording sites was performed during the dissections. At the time of HPLC-EC measurements, brain samples were sonicated in cold pH 4.1 mobile phase containing dihydroxybenzylamine ŽDHBA. as an internal standard. Samples were centrifuged at 16,000 = g for 10 min and 50 m l of supernatant was directly injected into the HPLC system coupled with dual-coulometric electrochemical detectors as previously described w16x. Tissue levels of DA, DOPAC, HVA, serotonin Ž5-HT. and 5-hydroxyindoleacetic acid Ž5-HIAA. were expressed as nanograms of analyte per gram wet weight of tissue Žngrg.. Sample size for each age group: young w n s 6x, middle-aged w n s 5x, old w n s 5x. 2.2.2. Statistical data analysis Data for monoamine tissue content and basal microdialysis measures were analyzed using One way analysis of variance ŽANOVA.. Analysis of locomotor activity and microdialysis data after D-amphetamine treatment utilized two way ANOVA with repeated measures. Data for the rod walk test were analyzed using a Kruskal–Wallis ANOVA on ranks and all pairwise comparisons utilized the Student–Newman–Keuls Method. Statistical results are reported within the text and figure legends.
3. Results 3.1. Rod walk test A rod walking test was used to study motoric performance of the young, middle-aged and old F344BNF1 rats. Scores for the rod walk test showed a significant Ž p - 0.05.
Fig. 1. Bar graph showing average rod walk test scores Ž"S.E.M.. for young Ž ns6., middle-aged Ž ns6., and old F344BNF1 rats Ž ns6.. Means were calculated using the best score from three trials for each animal. The following scale was used to determine the score for each test: 0—fall, 1—clasp, 2—all paws on top, 3—takes steps, 4—reaches platform. Kruskal–Wallis One way analysis of variance on ranks w H s 12.21, df s 2, p - 0.002x. ) p - 0.05, ŽStudent–Newman–Keuls Method..
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Fig. 2. Spontaneous and D-amphetamine-induced locomotor activity exhibited by young, middle-aged and old F344BNF1 rats. Spontaneous and D-amphetamine-induced locomotor activities were assessed on separate days. Locomotor activity was quantitated during a 2 h test session after habituating the animals to the test environment for 15 min. For D-amphetamine-induced locomotor activity, rats were injected with 5.0 mgrkg Ži.p.. D-amphetamine. Bars represent the average of each activity measure"S.E.M.; young Ž ns14., middle-aged Ž ns10., and old Ž ns13.. For statistical analysis, spontaneous and amphetamine-induced scores were designated as two levels of the independent variable DRUG. The AGE=DRUG interactions were significant for Horizontal Activity w F Ž2,37. s14.93, p- 0.001x, Total Distance w F Ž2,37. s 29.78, p- 0.001x, Movement Time w F Ž2,37. s 28.91, p- 0.001x, and Number of Movements w F Ž2,37. s12.71, p- 0.001x. ) p- 0.05, vs. young, )) p- 0.05 vs. middle-aged Žpost-hoc mean comparisons..
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age-related decline ŽFig. 1.. All young animals were able to reach the escape platform at least one time during the test trials. No middle-aged or old rats reached the escape platform during any test trials. Thus, the old and middleaged F344BNF1 rats exhibited diminished motor performance as compared to the young rats. 3.2. Locomotor actiÕity Naive young Ž n s 14., middle-aged Ž n s 10. or old Ž24 month old, n s 13. F344BNF1 rats were placed into Digiscan activity chambers, habituated to the test environment for 15 min, and then four components of locomotor activity were quantitated during a 2 h test period: total distance traveled during the test period, movement time Žmeasure of ambulation time., horizontal activity Žlight beam interruptions in a horizontal plane., average distance traveled per movement Žcentimetersrmovement.. Fig. 2 summarizes the results from these measures. All five measures of spontaneous locomotor activity in old rats were significantly lower than those observed for young rats. In old animals, two measures of ambulatory behavior, movement time and total distance traveled, showed significant 31 and 40% reductions, respectively, when compared to young animals. Interestingly, middle-aged rats showed decreases in only one spontaneous locomotor activity measure, horizontal activity. Middle-aged rats receiving D-amphetamine exhibited locomotor activity similar to that observed in young rats. The young, middle-aged, and old rats showed significant increases in all five locomotor activity measures ŽFig. 2. after administration of D-amphetamine Ž5.0 mgrkg, i.p... The magnitude of increase, however, was not the same for the three age groups; locomotor measures for young and middle-aged animals treated with D-amphetamine showed an average 3-fold increase above spontaneous levels, whereas locomotor measures for old F344BNF1 rats treated with D-amphetamine showed an average 2-fold increase above spontaneous levels. All five amphetamine-induce locomotor activity measures for old rats were significantly lower than corresponding measures in the young rats. These data indicate that both normal and pharmacologically-induced locomotor activity are reduced in aged rats when compared to young F344BNF1 rats. 3.3. Extracellular dopamine leÕels in the striatum: microdialysis Following behavioral tests, microdialysis was used to measure basal extracellular DA release in the striata of the young, middle-aged, and old rats ŽFig. 3.. Basal samples obtained from the striatum of middle-aged and old F344BNF1 rats contained significantly lower levels of DA as compared to young rats. Basal DOPAC levels showed an age-related decline Ž p - 0.05., and basal HVA was also significantly lower in the old vs. young animals Ž p - 0.05..
Fig. 3. Bar graph showing average basal release of dopamine in the striatum of young Ž ns14., middle-aged Ž ns9., and old Ž ns13. F344BNF1 rats. Three microdialysis samples of basal release were collected for each animal and an average value was calculated for each rat. Bars represent average values for each age group"standard error of the mean ŽS.E.M... One way ANOVA, w F Ž2,32. s 4.63x. ) p- 0.05 old vs. young, )) p- 0.05 middle-aged vs. young.
Thus, both basal DA and its metabolites were diminished in the old rats as compared to young animals. Changes in stimulated overflow of DA were examined by adding D-amphetamine Ž250 m M. to the perfusate for the duration of one sampling period Ž25 min.. This treatment produced an immediate overflow of DA in all age groups ŽFig. 4.. However, while young rats showed a robust increase in striatal DA levels immediately after D-amphetamine was added to the perfusate, with nearly a 25-fold increase in DA levels, the response in middle-aged or old rats was significantly lower. Extracellular levels of DA maintained a fairly stable rate of DA overflow during the 2 h sampling period in the young, and old rats. It was also noted that the addition of D-amphetamine to the perfusate of a probe implanted into one striatum did not alter the basal DA overflow in the contralateral striatum. The effect of D-amphetamine on striatal DOPAC levels in young rats is consistent with previous reports that D-amphetamine depresses extracellular levels of DOPAC w45x. Striatal DOPAC levels in middle-aged and old rats were similarly decreased by D-amphetamine with the exception that the magnitude of the response was diminished as compared to the responses recorded in the young rats ŽFig. 5.. Striatal HVA levels in old rats were also significantly lower than in young rats at all time points ŽFig. 6., while mean HVA levels for middle-aged rats were lower but not significantly different from those recorded in the young F344BNF1 rats. 3.4. Regional monoamine tissue content in young and aged F344BNF1 rats Following the behavioral and microdialysis studies, tissue was dissected from six different brain regions and monoamine levels and their metabolites were measured in each brain region of the young, middle-aged and old
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Fig. 4. Extracellular dopamine levels in the striatum of young, middle-aged, and old F344BNF1 rats before and after D-amphetamine stimulation. The first sample Žy50 min. was started 1.5 h after the microdialysis probe was inserted into the striatum; dialysate samples were collected every 25 min thereafter until a total of seven samples were obtained from each animal. At time 0 perfusate was switched to perfusate containing 250 m M D-amphetamine and then switched back to perfusate without D-amphetamine 25 min later. Circles represent the average amount of dopamine" S.E.M. detected in dialysate samples at each time point. Black circles, young rats Ž n s 14.; black triangles, middle-aged rats Ž n s 9.; white circles, old rats Ž n s 13.. Main effects of AGE w F Ž2,12. s 4.67, p - 0.05x and TIME w F Ž6,71. s 38.58, p - 0.001x were significant while the AGE = TIME interaction was not significant w F Ž12,71. s 0.50x. ) p - 0.05 vs. middle-age or old Žpost-hoc mean comparisons..
Fig. 5. Extracellular DOPAC levels in the striatum of young, middle-aged, and old F344BNF1 rats before and after D-amphetamine stimulation. The first sample Žy50 min. was started 1.5 h after the microdialysis probe was inserted into the striatum; dialysate samples were collected every 25 min thereafter until a total of seven samples were obtained for each animal. At time 0 perfusate was switched to perfusate containing 250 m M D-amphetamine and then switched back to perfusate alone 25 min later. Circles represent the average amount of dopamine" S.E.M. detected in dialysate samples at each time point. Black circles, young rats Ž n s 14.; black triangle, middle-aged rats Ž n s 9.; white circles, old rats Ž n s 13.. Main effects of AGE w F Ž2,12. s 15.71, p - 0.01x and TIME w F Ž6,71. s 8,73, p - 0.05x were significant while the AGE = TIME interaction was not significant w F Ž12,71. s 1.73x. ) p - 0.05 old vs. young Žpost-hoc mean comparisons..
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Fig. 6. Extracellular HVA levels in the striatum of young, middle-aged, and old F344BNF1 rats before and after D-amphetamine stimulation. The first sample Žy50 min. was started 1.5 h after the microdialysis probe was inserted into the striatum; dialysate samples were collected every 25 min thereafter until a total of seven samples were obtained for each animal. At time 0 perfusate was switched to perfusate containing 250 m M D-amphetamine and then switched back to perfusate only 25 min later. Circles represent the average amount of dopamine" S.E.M. detected in dialysate samples at each time point. Black circles, young rats Ž n s 14.; black triangle, middle-aged rats Ž n s 9.; white circles, old rats Ž n s 13.. Main effects of AGE w F Ž2,12. s 19.99, p - 0.05x was significant but not TIME w F Ž6,71. s 3.07x; the AGE = TIME interaction was not significant w F Ž12,71. s 2.48x. ) p - 0.05 old vs. young rats Žpost-hoc mean comparisons..
F344BNF1 rats ŽTable 1.. Interestingly, DA content in the midbrain ŽVTArsubstantia nigra. of the middle-aged and the old rats was also significantly lower than that measured in the substantia nigra of young animals. DA turnover
ratios wDOPACrDA, HVArDAx were significantly higher in the nucleus accumbens and ventral striatum of the old F344BNF1 rats as compared to young rats ŽTable 2.. Differences between DOPACrDA ratios in the substantia
Table 1 Neurochemical tissue content Žngrg tissue.
Medial prefrontal cortex
Nucleus accumbens
Ventral striatum
Dorsal striatum
Midbrain ŽSN q VTA.
Hippocampus
Age
Dopamine
DOPAC
HVA
NE
5-HT
5-HIAA
y m o y m o y m o y m o y m o y m o
44 " 13 27 " 10 29 " 7 1350 " 285 1178 " 295 848 " 211 6006 " 963 4616 " 554 3518 " 361) 6198 " 1347 4267 " 862 3475 " 615 447 " 111 182 " 18 ) ) 169 " 26 ) 8"2 3"1 2"1
161 " 65 89 " 24 62 " 10 1584 " 215 1759 " 243 1617 " 168 2284 " 288 2316 " 374 2448 " 207 2818 " 330 3484 " 321 3388 " 430 530 " 50 469 " 55 403 " 79 25 " 6 15 " 2 21 " 6
47 " 6 92 " 17 ) ) 57 " 4 425 " 59 483 " 72 449 " 47 801 " 75 827 " 115 814 " 68 959 " 194 998 " 102 802 " 88 169 " 15 155 " 18 137 " 25 9"2 8"2 8"4
171 " 12 177 " 41 135 " 19 727 " 123 742 " 86 679 " 80 150 " 36 143 " 18 117 " 27 38 " 13 56 " 11 42 " 14 228 " 21 173 " 15 153 " 13 ) 142 " 12 124 " 11 137 " 14
114 " 26 87 " 28 84 " 24 290 " 38 261 " 30 219 " 27 254 " 23 199 " 21 154 " 34 139 " 16 118 " 21 99 " 14 224 " 62 92 " 14 117 " 46 62 " 17 34 " 7 45 " 16
321 " 24 503 " 82 ) ) 325 " 32 ) ) ) 574 " 67 863 " 64 ) ) 715 " 53 431 " 24 583 " 62 ) ) 457 " 44 315 " 44 501 " 53 ) ) 318 " 37 ) ) ) 703 " 46 786 " 36 621 " 47 ) ) ) 384 " 19 465 " 27 ) ) 405 " 29
Pairwise comparisons: ) old vs. young, p - 0.05; m s middle-aged Ž n s 5., o s old Ž n s 5..
))
middle-aged vs. young, p - 0.05;
)))
old vs. middle-aged, p - 0.05; y s young Ž n s 6.,
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Table 2 Turnover ratios
Medial pre-frontal cortex
Nucleus accumbens
Ventral striatum
Dorsal striatum
Midbrain ŽSN q VTA.
Hippocampus
Age
DOPACrDA
HVArDA
ŽDOPACq HVA.rDA
5-HIAAr5-HT
y m o y m o y m o y m o y m o y m o
3.55 " 1.36 3.27 " 0.37 2.99 " 0.59 1.36 " 0.44 1.72 " 0.15 2.47 " 0.33 ) 0.41 " 0.05 0.47 " 0.10 0.70 " 0.10 ) 0.68 " 0.22 0.90 " 0.27 1.01 " 0.11 1.43 " 0.32 2.47 " 0.22 ) ) 2.41 " 0.34 ) 4.93 " 2.37 5.74 " 1.47 7.08 " 2.72
2.15 " 1.17 2.31 " 0.29 2.63 " 1.32 0.37 " 0.11 0.45 " 0.05 0.65 " 0.10 ) 0.15 " 0.02 0.17 " 0.03 0.23 " 0.03 ) 0.19 " 0.07 0.25 " 0.06 0.25 " 0.03 0.48 " 0.11 0.81 " 0.07 0.93 " 0.27 1.71 " 0.36 1.67 " 0.95 2.17 " 1.22
5.47 " 1.12 5.66 " 0.70 5.87 " 1.25 1.72 " 0.59 2.07 " 0.22 3.29 " 0.43 ) 0.56 " 0.06 0.64 " 0.12 0.94 " 0.12 ) 0.88 " 0.29 1.16 " 0.33 1.27 " 0.14 2.00 " 0.40 3.05 " 0.33 3.17 " 0.30 7.71 " 4.57 9.59 " 1.60 9.91 " 4.97
3.66 " 1.12 5.02 " 2.56 6.02 " 3.07 2.01 " 0.29 3.40 " 0.41) ) 3.43 " 0.26 ) 1.75 " 0.17 2.94 " 0.60 ) ) 3.36 " 0.41) 2.27 " 0.27 4.65 " 1.14 ) ) 3.27 " 0.36 5.58 " 5.20 10.26 " 5.59 13.36 " 6.53 8.09 " 6.11 16.22 " 4.59 15.52 " 8.74
Pairwise comparisons: ) old vs. young, p - 0.05;
))
middle-aged vs. young, p - 0.05; y s young Ž n s 6., m s middle-aged Ž n s 5., o s old Ž n s 5..
nigra of old vs. young rats were statistically significant Ž p - 0.05. while HVArDA ratios approached statistical significance Ž p - 0.06.. While no age-related differences in 5-HT content were observed in any of the brain regions examined, middle-aged F344BNF1 rats showed a significantly higher content of the 5-HT metabolite, 5-HIAA, in five of the six regions analyzed when compared to the same regions in young rats ŽTable 1.. The increase in 5-HIAA observed in two brain regions wmedial prefrontal cortex and dorsal striatumx of middle-aged rats appeared to be transient as old rats showed significantly lower 5-HIAA content while in the same regions the difference between old and young rats was not significant. Serotonin turnover w5-HIAAr5-HTx in the nucleus accumbens, ventral striatum, and dorsal striatum was significantly greater in middle-aged vs. young F344BNF1 rats, and this increase in turnover rate was maintained in the nucleus accumbens and ventral striatum of the old rats ŽTable 2..
4. Discussion In the present study, intracerebral microdialysis recordings from the striata of old F344BNF1 rats contained significantly lower basal levels of DA, DOPAC and HVA. Measurements of striatal DA, DOPAC, and HVA levels after D-amphetamine stimulation were also significantly lower in dialysis measurements from the striata of old rats as compared to the young animals. We also observed that old F344BNF1 rats showed significantly lower levels of both spontaneous and D-amphetamine-induced locomotor activity than those observed in young F344BNF1 rats.
Thus, functional measures of striatal DA were diminished in aged rats and these correlated with reduced motor performance in the aged animals. We measured five components of locomotor activity and noted significant age-related reductions in all five components. Our results are consistent with several other studies that have previously demonstrated age-related declines in motor function in aged rats. For example, age-related declines in the following motor tests have been reported: rod walking w13x, reaction time w4x, total activity measured during 15 min and 24 h periods w39x, swim performance w23,24x, incline screen, rod suspension, and rotorod tests w39x. It is important to point out that only one dose of amphetamine was used to characterized drug-induced activity in this study, and that a wider range of amphetamine doses are required to more fully characterize activity in the F344BNF1 rat strain. The decrease in basal and D-amphetamine-stimulated overflow of DA observed in the striatum of aged rats cannot be explained solely due to the assumption that there may be an age-related decline in the number of surviving DA neurons. First, age-related changes in the number of surviving neurons within the substantia nigra remains a controversial topic primarily because of the non-standardized stereologic techniques used to determine the numbers of cells in rat brain, and at this point there is no consensus on whether DA neurons decrease w10,36x or remain the same w29x during the normal aging process in rodents. Similarly, there is a lack of agreement regarding striatal DA tissue content in aged rats with some studies reporting decreases w8,15,18,23,25x while other studies report no age-related changes w5,10,13,28x. The differences in the reported values can be attributed to many factors
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including the measuring techniques, as well as the rat strain used in the studies. In this study we observed regional reductions of DA tissue content in the ventral striatum and midbrain of old rats. However, DA levels in the dorsal striatum of old rats were not significantly different than those measured in the same region of the young rat brain. Therefore, given the disparate findings of this study and the aforementioned studies, it remains difficult to determine if the observed decreases in extracellular DA are directly related to decreases in the number of surviving neurons. Second, there is evidence that decreasing the number of neurons within the substantia nigra alone does not necessarily result in decreased extracellular levels of DA within the rat striatum. In young adult rats, partial 6-hydroxydopamine lesions of the substantia nigra do not result in decreased levels of striatal DA as measured by microdialysis w34x, and it has been suggested that surviving DA neurons provide a compensatory response which restores extracellular DA levels to their pre-lesion state. The decrease in extracellular striatal DA observed in aged rats may be due to dopaminergic neurons that have become dysfunctional with age and exhibit deficiencies in controlling DA synthesis, overflow, and re-uptake. An example of dysfunctional neurons in aged Fisher 344 rats has been demonstrated by Friedemann and Gerhardt w13x. Our previous studies have shown age-dependent decreases in DA overflow and uptake within several different regions of the striatum without concurrent age-related alterations in tissue content of DA or DA metabolite. The results of this study suggest that functional changes of DA neurons can occur in aged rats while tissue content of DA remains unchanged. Similarly, Stamford w41x measured the rate of DA overflow in rat striatum using fast cyclic voltammetry and found significant differences in both the rate of overflow and the maximal amount of DA overflow in young adult vs. old rats. The regional differences we observed in this study in DA tissue may be related to diminished synthesis or storage of DA and not necessarily a loss of DA neurons. Therefore, it is important to consider that age-related changes in the ‘function’ of dopaminergic neurons may be just as important, if not more, than the actual ‘number’ of surviving neurons for controlling the amount of extracellular DA. While we have considered the possibility that dysfunctional dopaminergic neurons in aged rats might result in lower basal and D-amphetamine-stimulated levels of extracellular DA, we must also take into account the effect of halothane anesthesia on DA neurotransmission. Halothane on DA overflow has been characterized in several studies. However, in vivo measurements from rats under halothane anesthesia are not different from those in conscious animals. For instance, Fink-Jensen et al. w12x used microdialysis to measure the concentration of DA in the striatum of Sprague–Dawley rats Ž300 gm b.wt. and did not observe a significant difference in basal release between halothane anesthetized rats and conscious rats, nor was peak DA
concentration in the striatum of anesthetized and conscious rats different after the administration of D-amphetamine. Similarly, basal DA levels in the striatum of Sprague– Dawley rats Ž400 gm b.wt. were similar for halothanetreated rats with acute microdialysis probe implants and conscious rats with microdialysis probes implanted for a period of 1 day w31x. Miyano et al. w30x examined basal DA release in the striatum of Sprague–Dawley rats Ž250 gm b.wt. using various halothane concentrations and did not observe a significant increase in DA concentration until halothane concentration was increased to G 2%. While none of the aforementioned studies directly examined the variable of age on DA release in halothane anesthetized rats, the rats used in these studies were from a single strain ŽSprague–Dawley. that covered a moderate range of ages based upon their body weights. The results of these studies show a consistent finding that basal DA levels in the striatum of anesthetized or conscious rats are not significantly different at the level of anesthesia used in the present study. The age-related changes in striatal DA tissue content and DA turnover that occur in a dorsal-to-ventral direction are consistent with other reports of regional variations as a function of age. For example, Crawford and Levine w7x reported that D-amphetamine-induced c-Fos expression in the striatum of aged Fischer 344 rats shows a decline in the dorsal-to-ventral direction. Similarly, the ventral striatum of aged Fischer 344 rats exhibit significantly lower DA overflow and impaired DA uptake than that observed in young rats w13x. These data suggest that DA neurons in the ventral tegmental area or A10 region may be more susceptible to age-related decline in function than DA neurons in the A9 region of the substantia nigra. Several previous studies have shown that DA uptake mechanisms may be impaired in aged rodents w10,13,17,38,41x. It is well known that D-amphetamine produces a displacement of DA from its storage vesicles, and DA is released into the extracellular space by reverse transport by the DA transporter w14x. In the present study, D-amphetamine-evoked overflow of striatal DA was diminished in aged rats as compared to DA overflow recorded in young animals. This would suggest that DA neuronal uptake may be decreased in the aged brain. While we have not attempted to directly address this issue in our study, it is interesting to note that our microdialysis data may provide additional indirect evidence to support the hypothesis that DA uptake through DAT is impaired in aged rats. This can be observed by comparing the slopes of the D-amphetamine time course curves for young and old rats after peak DA overflow ŽFig. 4.. While young rats showed a sharp decline in DA levels, which is indicative of fast clearance and uptake rates, older rats showed a more gradual or slower clearance rate from the extracellular space than younger rats. If DA uptake mechanisms are impaired in aged rats, then one might predict a slower rate of DA clearance from striatal dialysate samples, as was
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observed in the present study and this has been observed previously w13,35x. Further studies are needed to more directly address the effects of aging on DA uptake in the striatum of aged F344BNF1 rats.
5. Conclusion We have used intracerebral microdialysis to measure extracellular levels of DA and DA metabolites in the striata of young, middle-aged and aged F344BNF1 rats and we have demonstrated an age-related reduction in striatal DA function. Basal DA levels were significantly lower in aged vs. young rat striatum, and DA metabolites were also found to be lower in the aged striatum. Similarly, Damphetamine-stimulated overflow of striatal DA appears to be impaired in older rats when compared to the more robust response observed in younger animals. Consistent with previous reports, we have observed that motor performance of old rats was deficient when compared to the performance of young rats. However, it still remains to be determined whether or not there is a direct relationshipŽs. between age-related reductions in striatal neurochemistry and reductions in motor activity.
Acknowledgements We would like to thank Shane Delinks for his assistance with the measurements of monoamines and metabolites in brain tissues. This research was supported by PHS grants NS09199 and AG06434 ŽGAG. and the NIA Pilot Program ŽDMY..
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w38x I. Shimizu, C. Prasad, Relationship between w 3 Hxmazindol binding to dopamine uptake sites and w 3 Hxdopamine uptake in rat striatum during aging, J. Neurochem. 56 Ž1991. 575–579. w39x E.L. Spangler, K.S. Waggie, J. Hengemihle, D. Roberts, B. Hess, D.K. Ingram, Behavioral assessment of aging in male Fischer 344 and Brown Norway rat strains and their F1 hybrid, Neurobiol. Aging 15 Ž1994. 319–328. w40x R.L. Sprott, Development of animal models of aging at the National Institute on Aging, Neurobiol. Aging 12 Ž1991. 635–638. w41x J.A. Stamford, Development and ageing of the rat nigrostriatal dopamine system studied with fact cyclic voltammetry, J. Neurochem. 52 Ž1989. 1582–1589. w42x J.E. Wallace, E. Krauter, B.A. Campbell, Motor and reflexive behavior in the aging rat, J. Gerontol. 35 Ž1980. 364–370. w43x H. Watanabe, Differential decrease in the rate of dopamine synthesis in several dopaminergic neurons of aged rat brain, Exp. Gerontol. 22 Ž1987. 17–25. w44x N. Zelnik, I. Angel, S.M. Paul, J.E. Kleinman, Decreased density of human striatal dopamine uptake sites with age, Eur. J. Pharmacol. 126 Ž1986. 175–176. w45x T. Zetterstrom, ¨ T. Sharp, C.A. Marsden, U. Ungerstedt, In vivo measurements of dopamine and its metabolites by intracerebral dialysis: changes after D-amphetamine, J. Neurochem. 41 Ž1983. 1769–1773.
Research report
Age-related decline in striatal dopamine release and motoric function in Brown NorwayrFischer 344 hybrid rats David M. Yurek
a,b,)
, Susan B. Hipkens a , Meleik A. Hebert c , Don M. Gash b , Greg A. Gerhardt
c
a Department of Surgeryr Neurosurgery, UniÕersity of Kentucky College of Medicine, Lexington, KY 40536, USA Department of Anatomy and Neurobiology, UniÕersity of Kentucky College of Medicine, Lexington, KY 40536, USA Departments of Psychiatry and Pharmacology, Neuroscience Training Program and the Rocky Mountain Center for Sensor Technology, UniÕersity of Colorado Health Sciences Center, DenÕer, CO 80262, USA b
c
Accepted 27 January 1998
Abstract The Brown NorwayrFischer 344 F1 hybrid rats ŽF344BNF1 . is a newer rat model and is emerging as an important rodent model of aging. In the present study we used motoric performance tests, intracerebral microdialysis, and neurochemical measures of postmortem brain tissue to investigate the effects of aging in young Ž4–5 months., middle-aged Ž18–19., and old Ž24–25 months. F344BNF1 hybrid rats. We observed that old F344BNF1 rats exhibited decreased motoric performance, and lower levels of spontaneous and Damphetamine-induced locomotor activity than those observed in young F344BNF1 rats. Microdialysis measures of extracellular basal levels of dopamine ŽDA., 3,4-dihydroxyphenylacetic acid ŽDOPAC., and 4-hydroxy-3-methoxyphenylacetic acid ŽHVA. were significantly diminished in the striata of the middle-aged and old rats as compared to levels in young animals. In addition, D-amphetamine-evoked overflow of DA was significantly decreased in the middle-aged and aged rat striatum as compared to DA overflow in young F344BNF1 rats. Studies of postmortem brain tissue showed that the changes in overflow of DA correlated with significantly lower DA tissue content in ventral striatum and midbrain. Moreover, both dopamine turnover ratios ŽDOPACrDA, HVArDA. and the serotonin turnover ratio Ž5-HIAAr5-HT. were significantly elevated in the ventral striatum and nucleus accumbens. The results of this study demonstrate a correlation between reductions in striatal DA neurochemistry and diminished motor function in aged F344BNF1 rats. q 1998 Published by Elsevier Science B.V. Keywords: Aging; 3,4-Dihydroxyphenylacetic acid; 4-Hydroxy-3-methoxyphenylacetic acid; D-amphetamine; Microdialysis; F344BNF1 rat
1. Introduction Aging in humans is associated with a decline in general motor skills w3x. The factors that produce motor dysfunction in aging are thought to be neurological in origin, and appear to be associated with a decrease in nigrostriatal dopaminergic activity w3,21x. Morphological studies indicate that DA neurons in the human midbrain may be lost at a rate of 4–6% per decade from the ages 20 to 90 during the normal course of aging w9,26x. Dopamine ŽDA. content in the caudate nucleus is decreased in aged humans w6x. Kish et al. w21x reported a 60% decline in striatal DA levels in normal subjects within the age range of 14 to 82 years. ) Corresponding author. Dept. of SurgeryrNeurosurgery, University of Kentucky Health Sciences Research Building, Lexington, KY 40536-0305, USA. Fax.: q1-606-323-6343; E-mail: [email protected]
Additional markers of the DAergic system have been found to decrease over the same time period. Levels of tyrosine hydroxylase ŽTH. mRNA in the substantia nigra, the rate limiting enzyme in DA synthesis, appear to decline steadily from young adulthood to old age w2x. Also, reductions of DA high-affinity uptake sites have been observed in aged humans w1,44x. Levels of DA transporter mRNA in the substantia nigra are also markedly decreased with age w2x. Clearly, changes in DA neuronal systems correlate with reduced motor skills in aged humans, and may be responsible for age-related reductions in motor abilities of humans. Rodent studies over the last 2 decades have produced equivocal results concerning changes in the nigrostriatal system with age. Dopamine synthesis w43x, catecholamine levels and catecholamine turnover w32x, tyrosine hydroxylase activity w27,37x, DA and DA metabolism w8,11x, DA
0006-8993r98r$19.00 q 1998 Published by Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 1 1 0 - 3
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release w13,35,41x, and DA receptor binding w37x all show significant declines in the striatum of aged rats when compared to similar measures in adult animals. In contrast, other studies report no changes in measures of nigrostriatal systems during aging w5,13,13,19,28,29x. These differences may be related to the animal models and techniques used to study the aged brain. Most of the aforementioned studies that examined DA and DA metabolites levels in the striatum of aged rodents have measured the total intracellular and extracellular catecholamine content within tissue samples. At the present time, few studies have addressed the dynamic release and metabolism of DA in the striatum of aged rats w20x. The objective of the present study was to use intracerebral dialysis as a means to investigate basal and stimulusevoked overflow of extracellular levels of DA and DA metabolites in the striatum of young and aged F344BNF1 rats, and to characterize locomotor activity and tissue neurotransmitter levels in the same animals. We chose the F344BNF1 rat in these studies because it is a model of aging that may have some advantages over other strains currently used for aging studies. This strain of rat is preferred over other strains because of its normal distribution of age-related pathologies occurring relatively late in life w40x. The aged F344BNF1 also has a low incidence of renal pathology, a common problem in aged Fischer 344 rats w22x. In addition, this hybrid strain does not have specific tumor susceptibilities that occur in some of the other rat models of aging. Preliminary studies which have examined age-related changes in motor function have indicated that the F344BNF1 rat exhibits a greater reduction of locomotor activity with age than does the Brown Norway or Fischer 344 strains w39x. Moreover, few systematic functional and behavioral studies of the DA neuronal system have been carried out in this relatively new rat model of aging.
2. Methods 2.1. Animals Male F344BNF1 rats ŽNIA, Charles River. were used in this study. Young Ž4–5 months old, n s 20., middle-aged Ž18–19 months old, n s 16., and old Ž24–25 months of age, n s 19. rats were housed in 26 = 48 = 20 cm acrylic cages with wood chips for bedding, and food and water available ad libitum. Animal cages were kept in a vivarium room and shelved within an environmental hoodrlaminar flow facility where temperature Ž23 " 18C., humidity Ž53 " 15%., and lightrdark cycle Ž12 h light:12 h dark. were closely controlled. Tissue samples and rod walk data were obtained from one cohort of rats at each age group: young w n s 6x, middle-aged w n s 6x, or old w n s 6x. Locomotor activity and microdialysis were obtained from separate cohort of rats: young w n s 14x, middle-aged w n s 10x, and
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old w n s 13x. All animal use procedures were approved by the University of Kentucky Animal Care and Use Committee, and were in strict accordance with the NIH Guide for the Care and use of Laboratory Animals. 2.1.1. Rod walk test Motor coordination and balance were assessed using a rod walking test w42x. The testing apparatus consisted of a 60 cm long wooden dowel of 5 cm in diameter that was grooved and suspended between two platforms 60 cm above a padded surface. Rats were placed in the center of the rod facing one of the platforms. Each rat was allowed three attempts to reach the escape platform, with a maximum time limit of 120 s. An additional rank score was also determined using the following scale: 0—fall, 1— clasp, 2—all paws on top, 3—takes steps, 4—reaches platform. Rats were assigned the best score from the three individual trials w13x. Sample size for each age group: young w n s 6x, middle-aged w n s 6x, old w n s 6x. 2.1.2. Locomotor actiÕity testing Locomotor activity was tested in Digiscan Animal Activity Monitors ŽOmnitech Electronics.. Each monitor contains an array of infrared sensors that are arranged in vertical and horizontal planes and are capable of measuring several components of animal activity simultaneously. This study focused on four measures of motor activity: horizontal activity, total distance, movement time Žambulation time., average distance per movement. Horizontal activity is defined as the number of infrared light beam interruptions that occur on the horizontal sensor during the test period. Total distance is defined as the total number of centimeters traveled by the animal during the test period. Movement time is defined as the time, in seconds, that the animal is ambulating; if the animal stops ambulating for longer than 1 s then this parameter is not incremented until the next ambulatory movement is made. Average distance per movement is measured in inches and is calculated as the average distance each animal traveled per ambulatory movement; an ambulatory movement is considered terminated if the animal remains non-ambulatory for longer than 1 s. Digiscan Activity monitors were housed directly in the animal room. All activity measurements were made during the dark phase of the animal’s diurnal cycle. Spontaneous locomotor activity was monitored by placing the animal inside the activity chambers, habituating the animals for 15 min, and then monitoring activity for a 2 h period. The D-amphetamine-induced locomotor activity was monitored similarly with the exception that the animals were administered D-amphetamine Ž5.0 mgrkg, i.p.. immediately after the habituation period. Spontaneous and D-amphetamineinduced locomotor activity were assessed in the same animals with a 1 week period intervening between each test. Sample size for each age group: young w n s 14x, middle-aged w n s 10x, old w n s 13x.
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2.2. Intracerebral microdialysis Each animal was anesthetized with a halothane–air mixture Ž1.0–1.5% halothane at 1.5 lrmin., placed in a stereotax, and maintained under halothane anesthesia for the duration of the microdialysis sampling period. Body temperature was maintained at 378C using a heating pad ŽCMA 150 Temperature Controller, Carneige Medicin.. Concentric dialysis probes Ž3.0 mm membranes, 0.5 mm diameter, CMAr12, Carneige Medicine. were used for all intracerebral dialysis studies. Dialysis probes were stereotactically lowered into each striatum Žbilateral, q1.0 mm A–P, "2.5 mm ML w33x. until the tip of the dialysis probe was 6.5 mm below the dura. The inlet of the dialysis probes were connected to a microinjection pump ŽCMA 100, Carneige Medicine. and perfusate w147 mM NaCl, 1.0 mM CaCl 2 , 3.9 mM KCl, pH s 6.0x was continuously pumped into the probe at a flow rate of 2.0 m lrmin. The first sample period began 1.5 h after the probe was stereotactically lowered into the brain. Striatal perfusates were collected every 25 min in microcentrifuge tubes containing 10 m l of 0.1 M perchloric acid, as a preservative, and each sample was subsequently stored at y808C. Sample size for each age group: young w n s 14x, middle-aged w n s 9x, old w n s 13x. For measurements of D-amphetamine-stimulated overflow of DA, the flow of perfusate into the left dialysis probe was switched from perfusate only to perfusate containing 250 m M D-amphetamine sulfate for 25 min, and then switched back to perfusate without D-amphetamine; right dialysis probe was not switched to D-amphetamine. Samples were assayed for levels of DA, DOPAC, and HVA using HPLC with electrochemical detection described in Ref. w18x. Compounds were separated on a 150 = 3 mm MD-150 column ŽESA.. Mobile phase wpH s 3.0, 75 mM sodium phosphate, 0.1 mM EDTA, 3.0 mM octyl sodium sulfate, and 10% acetonitrilex was pumped at a rate of 0.6 mlrmin. The HPLC system was coupled to dual-coulometric detectors ŽModel 5014B, ESA. with a pre-conditioning electrode set at y175 mV and the detection electrode set at 150 mV. Dialysate values were corrected for probe recoveries at 378C and are reported relative to 100% recovery. Probe recoveries were in the range of 9–15% " 1.4% for DA and 15–21% " 1.9% for DOPAC and HVA. 2.2.1. Determination of monoamine and monoamine metabolite leÕels in brain tissues Following the behavioral studies, rats were decapitated and their brains were quickly removed and placed in ice-cold saline. After cooling, tissue samples of the medial prefrontal cortex, nucleus accumbens, dorsal striatum, ventral striatum, midbrain Žsubstantia nigra and ventral tegmental area combined., and the hippocampus from the young, middle-aged and old rats were dissected, placed
into storage tubes and weighed, and stored at y708C. Anatomical verification of microdialysis probe recording sites was performed during the dissections. At the time of HPLC-EC measurements, brain samples were sonicated in cold pH 4.1 mobile phase containing dihydroxybenzylamine ŽDHBA. as an internal standard. Samples were centrifuged at 16,000 = g for 10 min and 50 m l of supernatant was directly injected into the HPLC system coupled with dual-coulometric electrochemical detectors as previously described w16x. Tissue levels of DA, DOPAC, HVA, serotonin Ž5-HT. and 5-hydroxyindoleacetic acid Ž5-HIAA. were expressed as nanograms of analyte per gram wet weight of tissue Žngrg.. Sample size for each age group: young w n s 6x, middle-aged w n s 5x, old w n s 5x. 2.2.2. Statistical data analysis Data for monoamine tissue content and basal microdialysis measures were analyzed using One way analysis of variance ŽANOVA.. Analysis of locomotor activity and microdialysis data after D-amphetamine treatment utilized two way ANOVA with repeated measures. Data for the rod walk test were analyzed using a Kruskal–Wallis ANOVA on ranks and all pairwise comparisons utilized the Student–Newman–Keuls Method. Statistical results are reported within the text and figure legends.
3. Results 3.1. Rod walk test A rod walking test was used to study motoric performance of the young, middle-aged and old F344BNF1 rats. Scores for the rod walk test showed a significant Ž p - 0.05.
Fig. 1. Bar graph showing average rod walk test scores Ž"S.E.M.. for young Ž ns6., middle-aged Ž ns6., and old F344BNF1 rats Ž ns6.. Means were calculated using the best score from three trials for each animal. The following scale was used to determine the score for each test: 0—fall, 1—clasp, 2—all paws on top, 3—takes steps, 4—reaches platform. Kruskal–Wallis One way analysis of variance on ranks w H s 12.21, df s 2, p - 0.002x. ) p - 0.05, ŽStudent–Newman–Keuls Method..
D.M. Yurek et al.r Brain Research 791 (1998) 246–256 249
Fig. 2. Spontaneous and D-amphetamine-induced locomotor activity exhibited by young, middle-aged and old F344BNF1 rats. Spontaneous and D-amphetamine-induced locomotor activities were assessed on separate days. Locomotor activity was quantitated during a 2 h test session after habituating the animals to the test environment for 15 min. For D-amphetamine-induced locomotor activity, rats were injected with 5.0 mgrkg Ži.p.. D-amphetamine. Bars represent the average of each activity measure"S.E.M.; young Ž ns14., middle-aged Ž ns10., and old Ž ns13.. For statistical analysis, spontaneous and amphetamine-induced scores were designated as two levels of the independent variable DRUG. The AGE=DRUG interactions were significant for Horizontal Activity w F Ž2,37. s14.93, p- 0.001x, Total Distance w F Ž2,37. s 29.78, p- 0.001x, Movement Time w F Ž2,37. s 28.91, p- 0.001x, and Number of Movements w F Ž2,37. s12.71, p- 0.001x. ) p- 0.05, vs. young, )) p- 0.05 vs. middle-aged Žpost-hoc mean comparisons..
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age-related decline ŽFig. 1.. All young animals were able to reach the escape platform at least one time during the test trials. No middle-aged or old rats reached the escape platform during any test trials. Thus, the old and middleaged F344BNF1 rats exhibited diminished motor performance as compared to the young rats. 3.2. Locomotor actiÕity Naive young Ž n s 14., middle-aged Ž n s 10. or old Ž24 month old, n s 13. F344BNF1 rats were placed into Digiscan activity chambers, habituated to the test environment for 15 min, and then four components of locomotor activity were quantitated during a 2 h test period: total distance traveled during the test period, movement time Žmeasure of ambulation time., horizontal activity Žlight beam interruptions in a horizontal plane., average distance traveled per movement Žcentimetersrmovement.. Fig. 2 summarizes the results from these measures. All five measures of spontaneous locomotor activity in old rats were significantly lower than those observed for young rats. In old animals, two measures of ambulatory behavior, movement time and total distance traveled, showed significant 31 and 40% reductions, respectively, when compared to young animals. Interestingly, middle-aged rats showed decreases in only one spontaneous locomotor activity measure, horizontal activity. Middle-aged rats receiving D-amphetamine exhibited locomotor activity similar to that observed in young rats. The young, middle-aged, and old rats showed significant increases in all five locomotor activity measures ŽFig. 2. after administration of D-amphetamine Ž5.0 mgrkg, i.p... The magnitude of increase, however, was not the same for the three age groups; locomotor measures for young and middle-aged animals treated with D-amphetamine showed an average 3-fold increase above spontaneous levels, whereas locomotor measures for old F344BNF1 rats treated with D-amphetamine showed an average 2-fold increase above spontaneous levels. All five amphetamine-induce locomotor activity measures for old rats were significantly lower than corresponding measures in the young rats. These data indicate that both normal and pharmacologically-induced locomotor activity are reduced in aged rats when compared to young F344BNF1 rats. 3.3. Extracellular dopamine leÕels in the striatum: microdialysis Following behavioral tests, microdialysis was used to measure basal extracellular DA release in the striata of the young, middle-aged, and old rats ŽFig. 3.. Basal samples obtained from the striatum of middle-aged and old F344BNF1 rats contained significantly lower levels of DA as compared to young rats. Basal DOPAC levels showed an age-related decline Ž p - 0.05., and basal HVA was also significantly lower in the old vs. young animals Ž p - 0.05..
Fig. 3. Bar graph showing average basal release of dopamine in the striatum of young Ž ns14., middle-aged Ž ns9., and old Ž ns13. F344BNF1 rats. Three microdialysis samples of basal release were collected for each animal and an average value was calculated for each rat. Bars represent average values for each age group"standard error of the mean ŽS.E.M... One way ANOVA, w F Ž2,32. s 4.63x. ) p- 0.05 old vs. young, )) p- 0.05 middle-aged vs. young.
Thus, both basal DA and its metabolites were diminished in the old rats as compared to young animals. Changes in stimulated overflow of DA were examined by adding D-amphetamine Ž250 m M. to the perfusate for the duration of one sampling period Ž25 min.. This treatment produced an immediate overflow of DA in all age groups ŽFig. 4.. However, while young rats showed a robust increase in striatal DA levels immediately after D-amphetamine was added to the perfusate, with nearly a 25-fold increase in DA levels, the response in middle-aged or old rats was significantly lower. Extracellular levels of DA maintained a fairly stable rate of DA overflow during the 2 h sampling period in the young, and old rats. It was also noted that the addition of D-amphetamine to the perfusate of a probe implanted into one striatum did not alter the basal DA overflow in the contralateral striatum. The effect of D-amphetamine on striatal DOPAC levels in young rats is consistent with previous reports that D-amphetamine depresses extracellular levels of DOPAC w45x. Striatal DOPAC levels in middle-aged and old rats were similarly decreased by D-amphetamine with the exception that the magnitude of the response was diminished as compared to the responses recorded in the young rats ŽFig. 5.. Striatal HVA levels in old rats were also significantly lower than in young rats at all time points ŽFig. 6., while mean HVA levels for middle-aged rats were lower but not significantly different from those recorded in the young F344BNF1 rats. 3.4. Regional monoamine tissue content in young and aged F344BNF1 rats Following the behavioral and microdialysis studies, tissue was dissected from six different brain regions and monoamine levels and their metabolites were measured in each brain region of the young, middle-aged and old
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Fig. 4. Extracellular dopamine levels in the striatum of young, middle-aged, and old F344BNF1 rats before and after D-amphetamine stimulation. The first sample Žy50 min. was started 1.5 h after the microdialysis probe was inserted into the striatum; dialysate samples were collected every 25 min thereafter until a total of seven samples were obtained from each animal. At time 0 perfusate was switched to perfusate containing 250 m M D-amphetamine and then switched back to perfusate without D-amphetamine 25 min later. Circles represent the average amount of dopamine" S.E.M. detected in dialysate samples at each time point. Black circles, young rats Ž n s 14.; black triangles, middle-aged rats Ž n s 9.; white circles, old rats Ž n s 13.. Main effects of AGE w F Ž2,12. s 4.67, p - 0.05x and TIME w F Ž6,71. s 38.58, p - 0.001x were significant while the AGE = TIME interaction was not significant w F Ž12,71. s 0.50x. ) p - 0.05 vs. middle-age or old Žpost-hoc mean comparisons..
Fig. 5. Extracellular DOPAC levels in the striatum of young, middle-aged, and old F344BNF1 rats before and after D-amphetamine stimulation. The first sample Žy50 min. was started 1.5 h after the microdialysis probe was inserted into the striatum; dialysate samples were collected every 25 min thereafter until a total of seven samples were obtained for each animal. At time 0 perfusate was switched to perfusate containing 250 m M D-amphetamine and then switched back to perfusate alone 25 min later. Circles represent the average amount of dopamine" S.E.M. detected in dialysate samples at each time point. Black circles, young rats Ž n s 14.; black triangle, middle-aged rats Ž n s 9.; white circles, old rats Ž n s 13.. Main effects of AGE w F Ž2,12. s 15.71, p - 0.01x and TIME w F Ž6,71. s 8,73, p - 0.05x were significant while the AGE = TIME interaction was not significant w F Ž12,71. s 1.73x. ) p - 0.05 old vs. young Žpost-hoc mean comparisons..
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252
Fig. 6. Extracellular HVA levels in the striatum of young, middle-aged, and old F344BNF1 rats before and after D-amphetamine stimulation. The first sample Žy50 min. was started 1.5 h after the microdialysis probe was inserted into the striatum; dialysate samples were collected every 25 min thereafter until a total of seven samples were obtained for each animal. At time 0 perfusate was switched to perfusate containing 250 m M D-amphetamine and then switched back to perfusate only 25 min later. Circles represent the average amount of dopamine" S.E.M. detected in dialysate samples at each time point. Black circles, young rats Ž n s 14.; black triangle, middle-aged rats Ž n s 9.; white circles, old rats Ž n s 13.. Main effects of AGE w F Ž2,12. s 19.99, p - 0.05x was significant but not TIME w F Ž6,71. s 3.07x; the AGE = TIME interaction was not significant w F Ž12,71. s 2.48x. ) p - 0.05 old vs. young rats Žpost-hoc mean comparisons..
F344BNF1 rats ŽTable 1.. Interestingly, DA content in the midbrain ŽVTArsubstantia nigra. of the middle-aged and the old rats was also significantly lower than that measured in the substantia nigra of young animals. DA turnover
ratios wDOPACrDA, HVArDAx were significantly higher in the nucleus accumbens and ventral striatum of the old F344BNF1 rats as compared to young rats ŽTable 2.. Differences between DOPACrDA ratios in the substantia
Table 1 Neurochemical tissue content Žngrg tissue.
Medial prefrontal cortex
Nucleus accumbens
Ventral striatum
Dorsal striatum
Midbrain ŽSN q VTA.
Hippocampus
Age
Dopamine
DOPAC
HVA
NE
5-HT
5-HIAA
y m o y m o y m o y m o y m o y m o
44 " 13 27 " 10 29 " 7 1350 " 285 1178 " 295 848 " 211 6006 " 963 4616 " 554 3518 " 361) 6198 " 1347 4267 " 862 3475 " 615 447 " 111 182 " 18 ) ) 169 " 26 ) 8"2 3"1 2"1
161 " 65 89 " 24 62 " 10 1584 " 215 1759 " 243 1617 " 168 2284 " 288 2316 " 374 2448 " 207 2818 " 330 3484 " 321 3388 " 430 530 " 50 469 " 55 403 " 79 25 " 6 15 " 2 21 " 6
47 " 6 92 " 17 ) ) 57 " 4 425 " 59 483 " 72 449 " 47 801 " 75 827 " 115 814 " 68 959 " 194 998 " 102 802 " 88 169 " 15 155 " 18 137 " 25 9"2 8"2 8"4
171 " 12 177 " 41 135 " 19 727 " 123 742 " 86 679 " 80 150 " 36 143 " 18 117 " 27 38 " 13 56 " 11 42 " 14 228 " 21 173 " 15 153 " 13 ) 142 " 12 124 " 11 137 " 14
114 " 26 87 " 28 84 " 24 290 " 38 261 " 30 219 " 27 254 " 23 199 " 21 154 " 34 139 " 16 118 " 21 99 " 14 224 " 62 92 " 14 117 " 46 62 " 17 34 " 7 45 " 16
321 " 24 503 " 82 ) ) 325 " 32 ) ) ) 574 " 67 863 " 64 ) ) 715 " 53 431 " 24 583 " 62 ) ) 457 " 44 315 " 44 501 " 53 ) ) 318 " 37 ) ) ) 703 " 46 786 " 36 621 " 47 ) ) ) 384 " 19 465 " 27 ) ) 405 " 29
Pairwise comparisons: ) old vs. young, p - 0.05; m s middle-aged Ž n s 5., o s old Ž n s 5..
))
middle-aged vs. young, p - 0.05;
)))
old vs. middle-aged, p - 0.05; y s young Ž n s 6.,
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Table 2 Turnover ratios
Medial pre-frontal cortex
Nucleus accumbens
Ventral striatum
Dorsal striatum
Midbrain ŽSN q VTA.
Hippocampus
Age
DOPACrDA
HVArDA
ŽDOPACq HVA.rDA
5-HIAAr5-HT
y m o y m o y m o y m o y m o y m o
3.55 " 1.36 3.27 " 0.37 2.99 " 0.59 1.36 " 0.44 1.72 " 0.15 2.47 " 0.33 ) 0.41 " 0.05 0.47 " 0.10 0.70 " 0.10 ) 0.68 " 0.22 0.90 " 0.27 1.01 " 0.11 1.43 " 0.32 2.47 " 0.22 ) ) 2.41 " 0.34 ) 4.93 " 2.37 5.74 " 1.47 7.08 " 2.72
2.15 " 1.17 2.31 " 0.29 2.63 " 1.32 0.37 " 0.11 0.45 " 0.05 0.65 " 0.10 ) 0.15 " 0.02 0.17 " 0.03 0.23 " 0.03 ) 0.19 " 0.07 0.25 " 0.06 0.25 " 0.03 0.48 " 0.11 0.81 " 0.07 0.93 " 0.27 1.71 " 0.36 1.67 " 0.95 2.17 " 1.22
5.47 " 1.12 5.66 " 0.70 5.87 " 1.25 1.72 " 0.59 2.07 " 0.22 3.29 " 0.43 ) 0.56 " 0.06 0.64 " 0.12 0.94 " 0.12 ) 0.88 " 0.29 1.16 " 0.33 1.27 " 0.14 2.00 " 0.40 3.05 " 0.33 3.17 " 0.30 7.71 " 4.57 9.59 " 1.60 9.91 " 4.97
3.66 " 1.12 5.02 " 2.56 6.02 " 3.07 2.01 " 0.29 3.40 " 0.41) ) 3.43 " 0.26 ) 1.75 " 0.17 2.94 " 0.60 ) ) 3.36 " 0.41) 2.27 " 0.27 4.65 " 1.14 ) ) 3.27 " 0.36 5.58 " 5.20 10.26 " 5.59 13.36 " 6.53 8.09 " 6.11 16.22 " 4.59 15.52 " 8.74
Pairwise comparisons: ) old vs. young, p - 0.05;
))
middle-aged vs. young, p - 0.05; y s young Ž n s 6., m s middle-aged Ž n s 5., o s old Ž n s 5..
nigra of old vs. young rats were statistically significant Ž p - 0.05. while HVArDA ratios approached statistical significance Ž p - 0.06.. While no age-related differences in 5-HT content were observed in any of the brain regions examined, middle-aged F344BNF1 rats showed a significantly higher content of the 5-HT metabolite, 5-HIAA, in five of the six regions analyzed when compared to the same regions in young rats ŽTable 1.. The increase in 5-HIAA observed in two brain regions wmedial prefrontal cortex and dorsal striatumx of middle-aged rats appeared to be transient as old rats showed significantly lower 5-HIAA content while in the same regions the difference between old and young rats was not significant. Serotonin turnover w5-HIAAr5-HTx in the nucleus accumbens, ventral striatum, and dorsal striatum was significantly greater in middle-aged vs. young F344BNF1 rats, and this increase in turnover rate was maintained in the nucleus accumbens and ventral striatum of the old rats ŽTable 2..
4. Discussion In the present study, intracerebral microdialysis recordings from the striata of old F344BNF1 rats contained significantly lower basal levels of DA, DOPAC and HVA. Measurements of striatal DA, DOPAC, and HVA levels after D-amphetamine stimulation were also significantly lower in dialysis measurements from the striata of old rats as compared to the young animals. We also observed that old F344BNF1 rats showed significantly lower levels of both spontaneous and D-amphetamine-induced locomotor activity than those observed in young F344BNF1 rats.
Thus, functional measures of striatal DA were diminished in aged rats and these correlated with reduced motor performance in the aged animals. We measured five components of locomotor activity and noted significant age-related reductions in all five components. Our results are consistent with several other studies that have previously demonstrated age-related declines in motor function in aged rats. For example, age-related declines in the following motor tests have been reported: rod walking w13x, reaction time w4x, total activity measured during 15 min and 24 h periods w39x, swim performance w23,24x, incline screen, rod suspension, and rotorod tests w39x. It is important to point out that only one dose of amphetamine was used to characterized drug-induced activity in this study, and that a wider range of amphetamine doses are required to more fully characterize activity in the F344BNF1 rat strain. The decrease in basal and D-amphetamine-stimulated overflow of DA observed in the striatum of aged rats cannot be explained solely due to the assumption that there may be an age-related decline in the number of surviving DA neurons. First, age-related changes in the number of surviving neurons within the substantia nigra remains a controversial topic primarily because of the non-standardized stereologic techniques used to determine the numbers of cells in rat brain, and at this point there is no consensus on whether DA neurons decrease w10,36x or remain the same w29x during the normal aging process in rodents. Similarly, there is a lack of agreement regarding striatal DA tissue content in aged rats with some studies reporting decreases w8,15,18,23,25x while other studies report no age-related changes w5,10,13,28x. The differences in the reported values can be attributed to many factors
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including the measuring techniques, as well as the rat strain used in the studies. In this study we observed regional reductions of DA tissue content in the ventral striatum and midbrain of old rats. However, DA levels in the dorsal striatum of old rats were not significantly different than those measured in the same region of the young rat brain. Therefore, given the disparate findings of this study and the aforementioned studies, it remains difficult to determine if the observed decreases in extracellular DA are directly related to decreases in the number of surviving neurons. Second, there is evidence that decreasing the number of neurons within the substantia nigra alone does not necessarily result in decreased extracellular levels of DA within the rat striatum. In young adult rats, partial 6-hydroxydopamine lesions of the substantia nigra do not result in decreased levels of striatal DA as measured by microdialysis w34x, and it has been suggested that surviving DA neurons provide a compensatory response which restores extracellular DA levels to their pre-lesion state. The decrease in extracellular striatal DA observed in aged rats may be due to dopaminergic neurons that have become dysfunctional with age and exhibit deficiencies in controlling DA synthesis, overflow, and re-uptake. An example of dysfunctional neurons in aged Fisher 344 rats has been demonstrated by Friedemann and Gerhardt w13x. Our previous studies have shown age-dependent decreases in DA overflow and uptake within several different regions of the striatum without concurrent age-related alterations in tissue content of DA or DA metabolite. The results of this study suggest that functional changes of DA neurons can occur in aged rats while tissue content of DA remains unchanged. Similarly, Stamford w41x measured the rate of DA overflow in rat striatum using fast cyclic voltammetry and found significant differences in both the rate of overflow and the maximal amount of DA overflow in young adult vs. old rats. The regional differences we observed in this study in DA tissue may be related to diminished synthesis or storage of DA and not necessarily a loss of DA neurons. Therefore, it is important to consider that age-related changes in the ‘function’ of dopaminergic neurons may be just as important, if not more, than the actual ‘number’ of surviving neurons for controlling the amount of extracellular DA. While we have considered the possibility that dysfunctional dopaminergic neurons in aged rats might result in lower basal and D-amphetamine-stimulated levels of extracellular DA, we must also take into account the effect of halothane anesthesia on DA neurotransmission. Halothane on DA overflow has been characterized in several studies. However, in vivo measurements from rats under halothane anesthesia are not different from those in conscious animals. For instance, Fink-Jensen et al. w12x used microdialysis to measure the concentration of DA in the striatum of Sprague–Dawley rats Ž300 gm b.wt. and did not observe a significant difference in basal release between halothane anesthetized rats and conscious rats, nor was peak DA
concentration in the striatum of anesthetized and conscious rats different after the administration of D-amphetamine. Similarly, basal DA levels in the striatum of Sprague– Dawley rats Ž400 gm b.wt. were similar for halothanetreated rats with acute microdialysis probe implants and conscious rats with microdialysis probes implanted for a period of 1 day w31x. Miyano et al. w30x examined basal DA release in the striatum of Sprague–Dawley rats Ž250 gm b.wt. using various halothane concentrations and did not observe a significant increase in DA concentration until halothane concentration was increased to G 2%. While none of the aforementioned studies directly examined the variable of age on DA release in halothane anesthetized rats, the rats used in these studies were from a single strain ŽSprague–Dawley. that covered a moderate range of ages based upon their body weights. The results of these studies show a consistent finding that basal DA levels in the striatum of anesthetized or conscious rats are not significantly different at the level of anesthesia used in the present study. The age-related changes in striatal DA tissue content and DA turnover that occur in a dorsal-to-ventral direction are consistent with other reports of regional variations as a function of age. For example, Crawford and Levine w7x reported that D-amphetamine-induced c-Fos expression in the striatum of aged Fischer 344 rats shows a decline in the dorsal-to-ventral direction. Similarly, the ventral striatum of aged Fischer 344 rats exhibit significantly lower DA overflow and impaired DA uptake than that observed in young rats w13x. These data suggest that DA neurons in the ventral tegmental area or A10 region may be more susceptible to age-related decline in function than DA neurons in the A9 region of the substantia nigra. Several previous studies have shown that DA uptake mechanisms may be impaired in aged rodents w10,13,17,38,41x. It is well known that D-amphetamine produces a displacement of DA from its storage vesicles, and DA is released into the extracellular space by reverse transport by the DA transporter w14x. In the present study, D-amphetamine-evoked overflow of striatal DA was diminished in aged rats as compared to DA overflow recorded in young animals. This would suggest that DA neuronal uptake may be decreased in the aged brain. While we have not attempted to directly address this issue in our study, it is interesting to note that our microdialysis data may provide additional indirect evidence to support the hypothesis that DA uptake through DAT is impaired in aged rats. This can be observed by comparing the slopes of the D-amphetamine time course curves for young and old rats after peak DA overflow ŽFig. 4.. While young rats showed a sharp decline in DA levels, which is indicative of fast clearance and uptake rates, older rats showed a more gradual or slower clearance rate from the extracellular space than younger rats. If DA uptake mechanisms are impaired in aged rats, then one might predict a slower rate of DA clearance from striatal dialysate samples, as was
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observed in the present study and this has been observed previously w13,35x. Further studies are needed to more directly address the effects of aging on DA uptake in the striatum of aged F344BNF1 rats.
5. Conclusion We have used intracerebral microdialysis to measure extracellular levels of DA and DA metabolites in the striata of young, middle-aged and aged F344BNF1 rats and we have demonstrated an age-related reduction in striatal DA function. Basal DA levels were significantly lower in aged vs. young rat striatum, and DA metabolites were also found to be lower in the aged striatum. Similarly, Damphetamine-stimulated overflow of striatal DA appears to be impaired in older rats when compared to the more robust response observed in younger animals. Consistent with previous reports, we have observed that motor performance of old rats was deficient when compared to the performance of young rats. However, it still remains to be determined whether or not there is a direct relationshipŽs. between age-related reductions in striatal neurochemistry and reductions in motor activity.
Acknowledgements We would like to thank Shane Delinks for his assistance with the measurements of monoamines and metabolites in brain tissues. This research was supported by PHS grants NS09199 and AG06434 ŽGAG. and the NIA Pilot Program ŽDMY..
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