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Effect of long-term caloric restriction on brain monoamines in aging male and female fischer 344 rats
(1989) 191--198
) R I C R E S T R I C T I O N ON BRAI ~E A N D F E M A L E FISCHER 34
FIOLSON a, P E T E R D U F F Y b and Natic of Reproductive and Developmental Toxicology and bOffice off Director, Dir !ical Research, Jefferson, Arkansas 72079 (U. S.A .) October 26, 1988) :eceived January 17th, 1989)
RY Lapresent study examines the changes in central monoamines mono~ a~ 44 n aged male and female rats after long-term caloric restrictio both sexes (n = 5 - - 1 0 / g r o u p ) were maintained on c one try J), Ls: ad libitum N I H 31 diet or 60% by weight of the ad aa lib. inta] ~ented with vitamins and minerals. Animals received 1these diet tge weeks until killed at 22.25 months of age. Caudate nucleus (CN), hypothalamus damus (HYPO), olfactory bulb (OB) and nucleus accumbens (NA) were assayed for content of norepinephrine (NE), dopamine (D (DA) and its metabolites (dihydroxy axyphenylacetic acid, D O P A C , and homovanillic acid, aci HVA) and serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) (5-HI using H P L C / E C . Relative.' to the ad lib. group, restricted rats of both sex show awed significant decreases in NE content antent in CN, H Y P O and OB. D A and 5-HT content were decreased significantly ina the CN and H Y P O . No significant changes were found fo in the levels of DA metabolites g: lites in all brain re~ions studied. While the 55-HIAA - H I A A level was significantly reduced in the H Y P O and N A o f the female restricted rats, it was increased severalfold in the OB of the male restricted icted animals. an These preliminary results suJggest that long-term caloric restriction alters brain m o n o a m i n e concentrations, ant effect which may in turn modify the normal rate of aging. .
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Key words: Chronic caloric restriction :riction; Central monoamines; Fischer 344 rats
int requests should be addressed. *To whom all correspondence and reprint Abbreviations: CN, caudate nucleus; DA, dopamine; DOPAC, dihydroxyphenylacetic acid; 5-HIAA, rotonin; HVA, homovanillic acid; HYPO, hypothalamus; NA, 5-hydroxyindoleacetic acid; 5-HT, serotonin; nucleus accumbens; NE, norepinephrine; le; OB, olfactory bulb; SPF, specific pathogen free. Printed and Published in Ireland
ories but adequate in all other ess~ han ad lib. feeding [1]. In recent y ndicate that caloric restriction not the species and reduces the rate lters the time to occurrence of vi~ :luding cancer, arthritis, renal dis~ hich age at a much slower rate in ed animals, include the immune system [5,6], eye lens len~, protein, d the aging of collagen [7]. Functional parameters such : as n and the response to hormones [8] as well as behav behavioral am ~dents. s [9] also appear to age more slowly in restricted rodei res present study was designed to determine whether caloric c~ would also affect monoamine concentrations in various v~ br~ a assess the alteration of DA, 5-HT and their metabolites, metal [ t I A A as well as NE in response to caloric restriction,, we meas e neurotransmitters in those brain areas involved iin feedin~ ag H Y P O , OB, N A and CN of male and female Fischer Fiscb 344 rat
nts erses
ing geeo-
iiet ta-
ine tor 19 In VA 'els ty,
[ALS AND METHODS
AdultIt male and female Fischer 344 rats were raised at tthe National Center for at 23°C _+ logical Research in a specific pathogen free (SPF) environment e' Toxicolc with lights on at 0600 h. 1 °C andd were maintained at a 12/12 h, light/dark cycle w Animalss were divided at 14 weeks of age into ad lib. controls control, and a caloric restricted hat received 60070 by weight of the food consumed byy the ad lib. rats. A d lib. group that intake was Nas not corrected for spillage, but the feeding regime employed in the present Cas effective in maintaining restricted body weight at a below 65°7o of ad lib. study was h r n n ~ h n n t tthe h e dduration n r a t i oon n ooff the t h e sstudy. tudy_ W ater w a s rprovided ad lib. to both Water was weights tthroughout nent. All rats were fed a standard NIH-31 diet with groups throughout the experiment. lented for the restricted subjects. The restricted ration vitamins and minerals supplemented was given and consumed daiby at l l 0 0 h. In contrast, ad lib. rats consumed the majority of their food at 2300 h, roughly 5 h after the onset of the dark phase. Animals were housed individually in plastic cages with metal tops and hardwood chip bedding. Animals at the age ofF 21 months were transferred from the SPF barrier to the testing laboratory where theey were maintained under similar environmental conaperiod was shifted by 12 h for both groups and the ditions. At that time the photor rats were allowed to adapt to the new light/dark cycle for an additional 35 days before time of killing. This adaaptation period (35 days) has been shown to be suffiological cient to normalize all the physiologica ical parameters to preshift levels [10]. In order to en during this 35-day period, food was presented to synchronize the feeding regimen
k phase, so that both control and the same time of the day (1100 h).
"ats
2.25 months by decapitation in c~ zed All 1500 (7 h after the onset of the dl ar any abnormal organ growth c cal The brain was quickly removed [A, )idly on a cold surface according 'ied [ of Glowinski and Iversen [11]. Brain regions were weighed w ar ely ,eron solid CO 2, then stored at - 70°C until a neurotransmitter neurotr~ using high-performance liquid chromatography ( ,ith brain samples were place .'hemical detection [12]. Frozen -ml an loft polyproplene microtubes and thoroughly homoggenized fc Sot nic cell disrupter (Heat Systems, model W-350, Branson Bra O., mtrol dial settir :y, CT) at 40°70 pulsed power, with an output control, tissues were homogenized in 50--100/~I of ice-cold mobile p 55) Ling 650 pg//al o f dihydroxybenzylamine (DHBA) as a an int¢ rd. ere s were then centrifuged (20 000 g, 30 rain, 4°C) and ant the sup a RC ane ed and filtered through a microfilter tube loaded with w tri~lytical System, West Lafayette, IN). The microfilter tubes wl 4000 g, 5 min, 4°C) and aliquots (5--10 ~I) of tissue tissl sample were injected directly into the H P L C . Separation ~.ion o f the monoamines and their metabolites Altex Scientific Inc., BerkeThe H P L C system consisted of a B-100 Altex p u m p (Alte 9article size, 7.5 cm × 4.6 ley, CA)L) connected to a supercosil LC-18 column (3 gm par m m I.D., Supelco, Inc., Bellofante, PA) with stainless steel sample injectors any sample contamequipped .~d wih a 20/al sample loop. To protect the column from fr, u n r ~ r t ~ h l r n n o l l a r c l n ~ t i c ~ r t ~ i t h I O ,,rt~ N l ~ q ] r ~ v , inants, a precolumn guard packed tcked with 10 /am ODS, reversed phase was placed between the analytical columnI and the sample injector. An. amperometric electroioanalytical System Inc., West Lafayette, IN) with chemical detector (LC-4A, Bioanal ]e was used at a voltage setting of + 0.8 V vs. silver : glassy carbon working electrode silver chloride reference electrode. ade. The stock mobile phase was prepared in 4.0 1 (to:ion of the previously described method of Lin and tal vol.) by a slight modification Blank [12]. It contained 0.1 M citric acid monohydrate, 0.05 mM Na 2 E D T A and 0.4 m M octylsodium sulfate dissolved lved in distilled deionized water. This stock solution was tightly covered and kept at 4°C and an aliquot was taken as needed (usually within the same week). The workin ,orking mobile phase was prepared daily by filtering n, then adding 100 ml (10O7o) of acetonitrile ( H P L C 900 ml from the stock solution. grade) and 0.9 ml (0.09°70) o ff diethylamine. The entire mobile phase was slowly stirred and the p H was adjusted ~d to 2.55 _+ 0.01 using solid sodium hydroxide then
triction on Monoamine Levels date Nucleus)
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',d under vacuum for 20--30 min. The mobile phase was recycled overnight degassed for 16--20 -20 h in order to stabilize the analytical column and to obtain a stable baseline. Flow ow rate was 0.8 m l / m i n throughout the procedure. The T electric output of the detectorr, representing the peaks of different compounds, was quantitated using a n t e ~ r a t n r ((Hewlett-Packard l - l e w l e t t - P a c,'kard, k a r d _ Avondale, A v n n d a l e . PA). PAL 3380 A iintegrator ished by obtaining the percent (ratio) of peak areas of A standard curve was established 5 concentrations of each c o mLpOUnd (313--2500 pg//al) to that of D H B A . Tissue sample quantitation was obtained ned by comparing the ratio to the standard curve values. The final concentration off monoamines mono~ noamines and their metabolites was expressed on weight (g). Student's t-test was used for significant the basis of the original tissue differences between ad lib. andJ restricted groups. Statistical analysis was conducted separately for each sex. RESULTS
The final body weight at thee time of sacrifice was 249 ___ 22 and 459 ___ 23 g for the male restricted and ad lib. animals, respectively and 167.0 + 21 and 322.5 _+ 54.0 g for the female restricted and ad lib. rats, respectively.
3mine Levels
Male, Restricted Male, ad libitum ~
Female, Restricted
i
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Fig. 1. The te conc~ concentration of monoamines and their metabolites in the caudate caud~ nucleus (Fig. l-A), hypothalamus (Fig. I-B), olfactory bulb (Fig. l-C) and nucleus accumbens (Fig. l-D) 1 of 22.25-month-old male le Fischer 344 rats. Animals were maintained on an ad lib. or restricted r diet for 18 months and female :rifice. Each value is the mean _+ S.E.M. Number above each bbar represents the number of before sacrifice. rats/group. Significant differences between ~¢een restricted and adlib, in both sexes are denoted by * P < 0.05, **P< 0.01.
The effects of long-term caloric oric restriction on the concentration of monoamines and their metabolites in the CN N o f male and female Fischer 344 rats are shown in Fig. 1-A. NE, DA, 5-HT andI H V A were significantly reduced in the calorically restricted groups. NE concentration 'ation in this region was reduced by 75070 (male) and 90°70 (female) in the caloric restricted ricted animals as compared to the ad lib. control rats of the same sex. D A and 5-HT IT concentrations were reduced by 56°7o and 62070, respectively, in the CN o f male role restricted rats. The concentrations of various neurotransmitters in the H Y P O) are presented in Fig. 1-B. In the male rats, only 5H T was significantly reduced (by 36070 by 36070) 5070) in the caloric restricted animals, when compared to the ad lib. rats. In contra~ ntrast, the female rats of the caloric restricted group
(35%), DA (33%) D O P A C (23% I to ad lib. control rats. ~oth sexes of the caloric restricted J 42% (female) as compared to the ificant sex difference in the DA females of both groups had lower 'actory bulb of the male resticted r trois. There was a significant incre ale restricted animals as compared s or to the Iemale of both experimental ~erimental groups. effect of caloric restriction on monoamine concentrations concenm in in Fig. 1-D. The concentrations of 5-HT and 5-HIAA 5-HI• in thL, restricted rats were significantly reduced by 37% an and 55%, r 'ed to the ad lib. animals.
%) fifals ~C )ns
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~ION
preliminary study suggests that long-term caloric restrictio tly the content of various neurotransmitter in several brain re ale female Fischer 344 rats. In all brain regions studied, stuc diet on .~d to decrease the concentration of some of the monoar leir lites with a single exception of 5 - H I A A in the OB of the male restricted rats where the level was greatly increased. content in the Ther~'e are many reports indicating that diet can alter monoamine mo tryptophan availability is the or the serotonergic system, it is well known that trypt CNS. For in brain 5-HT levels rate-limitin fiting step in 5-HT synthesis. The substantial reduction reducti in caloric ric restricted rats found in this study could be a consequence c of reduced tryptophan available for dietary tryptophan, which in turn affected the amount of t~ brain uptake. On the other hand, some other types of diet di~ restriction have been reportedd to increase 5-HT concentration in the brains of undernourished t animals [13--17], perhaps due to reduced tryptophan competitive binding by serum Lncement nent of tryptophan uptake by the CNS [16--18]. albumins and subsequent enhancemen The present study showed that the decrease in the 5-HT level in both the HYPO and tease in the level of its metabolite 5-HIAA, suggesting NA was accompanied by a decrease a reduction in 5-HT synthesis. On the other hand, elevated 5-HIAA concentration in a t suggests that 5-HT is metabolized more rapidly in the OB of the male restricted rat this region, resulting in deple.~tion of 5-HT. These findings suggest that dietary restriction has complex effects on CNS 5-HT contents, effects which are regionally nd on the precise nature, onset and duration of the specific, and doubtlessly depend nutritional deprivation employed. Similarly, dietary restrictiona may well reduce catecholcomine synthesis. Tyrosine hydroxylase is the rate-limitingg enzyme in the biosynthetic pathway which converts
hat mation o f NE. Recently, it has bc lch osine hydroxylase activity in rat b ith ing effect upon neurotransmitter : ely n the sequence, such as NE being Lse in this study, where NE levels ted ntial effect is in agreement with a~ ort H Y P O of food-deprived rats [20] duced alterations in CNS monoamJ [ter mic 5-HT, NE and DA are knox~ ice y function and hence the entire hormonal mileau [21 21--24]. T1 Lin alamic monoamines found in this study could thus have a pn act he endocrine system. It has recently been reporte ~orted that c ary ion increases basal glucocorticoid levels [25], a findin~ g replicat ~o'unpublished observation). Changes in corticosterom 9ne levels, i: for ime or over the life span, could be expected to have a substant na nge of biological variables, perhaps including tg the aging agir proces~ tmmary, the present study suggests that long-term caloric cal restr ter rochemical balance in the brain. This effect occurred despite t of ogical mechanisms that protect the brain against shifts shi in the or punt of important nutrients. Conclusions from these th preli 1Its be made very cautiously until further evidence is ob! obtained on md n of caloric or diet restriction on other factors that regulate normal neurotransmitters [tters levels. These including peripheral and central concentrations c of amino acids such uch as tyrosine and tryptophan, changes in the Va~ of the rate-limiting enzymes:s of monoamines biosynthesis, the activity of metabolizing metab, enzymes and the turnover ~'r rate of these monoamines under such experimenta ~erimental conditions. It will also be necessa ;ssary to clearly distinguish between the effects of short-term (acute) and long-term rm (chronic) food deprivation on CNS monoamines. ACKNOWLEDGMENTS
The authors express their a ppreciation to Miss Kellye Walker and Ms. Barbara Jacks for preparing this manuscrt ascript. This work was supported by the Oak Ridge Associated Universities P r o g r atm m at the National Center for Toxicological Research (NCTR). REFERENCES 1 2 3
C.M. McCay, M.F. Crowell andI L.A. Maynard, The effect o f retarded growth upon the length of life span and upon the ultimate bod' ody size. J. Nutr., 10 (1935) 63--80. ation and life prolongation. In: C.E. Finch and L. Hayflick (eds.) G.A. Sacher, Life table modificat Handbook o f the Biology o f Aging, ng, Van Nostrand Reinhold, New York, 1977, 582--638. E.J. Masoro, Nutrition and aginlg - - a current assessment. J. Nutr., 115 (1985) 842--848.
xperimental manipulation of aging by di~ [e span and spontaneous cancer incidenc Science, 215 (1982) 1415--1418. -DeLima, M. Mathies and G.S. Smith, I_ .~: response to sheep red blood cells and to
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ary ary ItS.
logical assays for biological age in mice: r~ :ol9t. Aging Res., 9 (1984) 245--251. r, Effect of diet restriction on some bioch( ers 983) 944--950. ngler, J.R. Freeman and R.L. Walford, I ion fits learning and motor performance of aged mice• J. Gerontol., 42 4~ (1987) 78of • Duffy, R.J. Feuers, J.E.A. Leakey, K.D. N a k a m u r a , A. Turturro Turtur and R.V metab nic caloric restriction on the physiological variables related to energy en ale her 344 rat. Mech. Ageing Dev., 48 (1989) 117-- 133. lowinski and L.L. Iversen, Regional studies of catecholamines in the rat brai ~siof ~H-norepinephrine, 3H-dopamine and 3H-dopa in various reg ions of the ro~., 13 (1966) 655--669. Lin and C.L. Blank, Utilizing 3 /am reverse phase columsn for LCEC dete ~io: amines. Current Separation, 5 (1983) 3--6. Sobotka, M.P. Cook and R.E. Brodie, Neonatal malnutrition: tneurochemi md tvioral manifestations. Brain Res., 65 (1974) 443--457. lisatomi and Y. Niiyama, Effects of postnatal undernutrition on the catecho roa content of suckling rat brain. J. Nutr. Sci. Vitaminol., 26 (1980) 279--292. ?. Stern, M. Miller, W.B. Forbes, P.J. Morgane and O. Resnick,, Ontogeny ( o rioc amines in various parts of the brain and in peripheral tissues in normal tI I U l l | l a l aand Ll~ VlvtbLtl l l l a l I t V U lu- r :1rats. Expt. Neurol., 49 (1975) 314--326. M . MVliller iller and O. Resnick, Tryptophan availability: the importance mrtance of o prepartum and postpartum dietar lry protein on brain indolamine metabolism in rats• Expt. Neurol., 67(1980) 298--314. M. Miller diller, J.P. Leahy, W . C . Stern, P.J. Morgane and O. Resnick, Tryyptophan availability: relation to elevated evated brain serotonin in developmentally protein-malnourished )rotein-malnourished :rats. Exp. NeuroL. 57 (1977) 142--157. :tlon on serotonin metabolism in S. Kohsaka ohsaka, K. T a k a m a t s u and Y. Tsukada, Effect of food restrictiot rat brain• ~rain. Neurochem. Res., 5 (1980) 69--93. feeding on neuroir E.D.• London, S.B. Waller, A.T. Ellis and D.K. Ingram, Effects of intermittent chemical nical markers in aging rat brain. Neurobiol. Aging, 6 (1985) 199--204• 199--2 ,othalamic norepinephrine by food depriS,D.• Glick, D.H. Waters and S. Milloy, Depletion of hypothalamic n vation and interaction with d-am phetamine. Res. Communs. Chem. Pathol. Pharmacol., 6 (1973) 775--778. mines and A C T H secretion. In Ganang and Martini teds.), Fron+ G.R. Van Loon, Brain catecholamines ord University Press, New York, 1973, pp. 209--247. tiers in Neuroendocrinology, Oxford G.R. Van Loon, U. Scapagnini, G.P. Moberg and W.F. Ganong, Evidence of central adrenergic retion in the rat. EndocrinoL, 89 (1971) 1464-- 1469. neuronal inhibition of A C T H secretion R.M. Eisenberg, Further evidencee of a central alpha-adrenergic inhibitory influence on the hypothalamo-pituitary-adrenal axis in thee rat. Neuroendocrinology, 17 (1975) 154-- 166. M.T. Jones, E.W. Hillhouse and:1 J. Burden, Effect of various putative neurotransmitters on the ing h o r m o n e from the rat hypothalamus in vitro - - A model of the secretion of corticotrophin-releasin Fdocrinol., 69 (1976) 1--10. neurotransmitters involved• J. Endocrinol. A. Armario, J.L. Montero and T. Jolin, Chronic food restriction and the circadian rhythms t>i pituitary-adrenal hormones, growth h o r m o n e and thyroid-stimulating hormone. Ann. Nutr+ Metab., 31 (1987) 81--87.
) R I C R E S T R I C T I O N ON BRAI ~E A N D F E M A L E FISCHER 34
FIOLSON a, P E T E R D U F F Y b and Natic of Reproductive and Developmental Toxicology and bOffice off Director, Dir !ical Research, Jefferson, Arkansas 72079 (U. S.A .) October 26, 1988) :eceived January 17th, 1989)
RY Lapresent study examines the changes in central monoamines mono~ a~ 44 n aged male and female rats after long-term caloric restrictio both sexes (n = 5 - - 1 0 / g r o u p ) were maintained on c one try J), Ls: ad libitum N I H 31 diet or 60% by weight of the ad aa lib. inta] ~ented with vitamins and minerals. Animals received 1these diet tge weeks until killed at 22.25 months of age. Caudate nucleus (CN), hypothalamus damus (HYPO), olfactory bulb (OB) and nucleus accumbens (NA) were assayed for content of norepinephrine (NE), dopamine (D (DA) and its metabolites (dihydroxy axyphenylacetic acid, D O P A C , and homovanillic acid, aci HVA) and serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) (5-HI using H P L C / E C . Relative.' to the ad lib. group, restricted rats of both sex show awed significant decreases in NE content antent in CN, H Y P O and OB. D A and 5-HT content were decreased significantly ina the CN and H Y P O . No significant changes were found fo in the levels of DA metabolites g: lites in all brain re~ions studied. While the 55-HIAA - H I A A level was significantly reduced in the H Y P O and N A o f the female restricted rats, it was increased severalfold in the OB of the male restricted icted animals. an These preliminary results suJggest that long-term caloric restriction alters brain m o n o a m i n e concentrations, ant effect which may in turn modify the normal rate of aging. .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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¢~_
Key words: Chronic caloric restriction :riction; Central monoamines; Fischer 344 rats
int requests should be addressed. *To whom all correspondence and reprint Abbreviations: CN, caudate nucleus; DA, dopamine; DOPAC, dihydroxyphenylacetic acid; 5-HIAA, rotonin; HVA, homovanillic acid; HYPO, hypothalamus; NA, 5-hydroxyindoleacetic acid; 5-HT, serotonin; nucleus accumbens; NE, norepinephrine; le; OB, olfactory bulb; SPF, specific pathogen free. Printed and Published in Ireland
ories but adequate in all other ess~ han ad lib. feeding [1]. In recent y ndicate that caloric restriction not the species and reduces the rate lters the time to occurrence of vi~ :luding cancer, arthritis, renal dis~ hich age at a much slower rate in ed animals, include the immune system [5,6], eye lens len~, protein, d the aging of collagen [7]. Functional parameters such : as n and the response to hormones [8] as well as behav behavioral am ~dents. s [9] also appear to age more slowly in restricted rodei res present study was designed to determine whether caloric c~ would also affect monoamine concentrations in various v~ br~ a assess the alteration of DA, 5-HT and their metabolites, metal [ t I A A as well as NE in response to caloric restriction,, we meas e neurotransmitters in those brain areas involved iin feedin~ ag H Y P O , OB, N A and CN of male and female Fischer Fiscb 344 rat
nts erses
ing geeo-
iiet ta-
ine tor 19 In VA 'els ty,
[ALS AND METHODS
AdultIt male and female Fischer 344 rats were raised at tthe National Center for at 23°C _+ logical Research in a specific pathogen free (SPF) environment e' Toxicolc with lights on at 0600 h. 1 °C andd were maintained at a 12/12 h, light/dark cycle w Animalss were divided at 14 weeks of age into ad lib. controls control, and a caloric restricted hat received 60070 by weight of the food consumed byy the ad lib. rats. A d lib. group that intake was Nas not corrected for spillage, but the feeding regime employed in the present Cas effective in maintaining restricted body weight at a below 65°7o of ad lib. study was h r n n ~ h n n t tthe h e dduration n r a t i oon n ooff the t h e sstudy. tudy_ W ater w a s rprovided ad lib. to both Water was weights tthroughout nent. All rats were fed a standard NIH-31 diet with groups throughout the experiment. lented for the restricted subjects. The restricted ration vitamins and minerals supplemented was given and consumed daiby at l l 0 0 h. In contrast, ad lib. rats consumed the majority of their food at 2300 h, roughly 5 h after the onset of the dark phase. Animals were housed individually in plastic cages with metal tops and hardwood chip bedding. Animals at the age ofF 21 months were transferred from the SPF barrier to the testing laboratory where theey were maintained under similar environmental conaperiod was shifted by 12 h for both groups and the ditions. At that time the photor rats were allowed to adapt to the new light/dark cycle for an additional 35 days before time of killing. This adaaptation period (35 days) has been shown to be suffiological cient to normalize all the physiologica ical parameters to preshift levels [10]. In order to en during this 35-day period, food was presented to synchronize the feeding regimen
k phase, so that both control and the same time of the day (1100 h).
"ats
2.25 months by decapitation in c~ zed All 1500 (7 h after the onset of the dl ar any abnormal organ growth c cal The brain was quickly removed [A, )idly on a cold surface according 'ied [ of Glowinski and Iversen [11]. Brain regions were weighed w ar ely ,eron solid CO 2, then stored at - 70°C until a neurotransmitter neurotr~ using high-performance liquid chromatography ( ,ith brain samples were place .'hemical detection [12]. Frozen -ml an loft polyproplene microtubes and thoroughly homoggenized fc Sot nic cell disrupter (Heat Systems, model W-350, Branson Bra O., mtrol dial settir :y, CT) at 40°70 pulsed power, with an output control, tissues were homogenized in 50--100/~I of ice-cold mobile p 55) Ling 650 pg//al o f dihydroxybenzylamine (DHBA) as a an int¢ rd. ere s were then centrifuged (20 000 g, 30 rain, 4°C) and ant the sup a RC ane ed and filtered through a microfilter tube loaded with w tri~lytical System, West Lafayette, IN). The microfilter tubes wl 4000 g, 5 min, 4°C) and aliquots (5--10 ~I) of tissue tissl sample were injected directly into the H P L C . Separation ~.ion o f the monoamines and their metabolites Altex Scientific Inc., BerkeThe H P L C system consisted of a B-100 Altex p u m p (Alte 9article size, 7.5 cm × 4.6 ley, CA)L) connected to a supercosil LC-18 column (3 gm par m m I.D., Supelco, Inc., Bellofante, PA) with stainless steel sample injectors any sample contamequipped .~d wih a 20/al sample loop. To protect the column from fr, u n r ~ r t ~ h l r n n o l l a r c l n ~ t i c ~ r t ~ i t h I O ,,rt~ N l ~ q ] r ~ v , inants, a precolumn guard packed tcked with 10 /am ODS, reversed phase was placed between the analytical columnI and the sample injector. An. amperometric electroioanalytical System Inc., West Lafayette, IN) with chemical detector (LC-4A, Bioanal ]e was used at a voltage setting of + 0.8 V vs. silver : glassy carbon working electrode silver chloride reference electrode. ade. The stock mobile phase was prepared in 4.0 1 (to:ion of the previously described method of Lin and tal vol.) by a slight modification Blank [12]. It contained 0.1 M citric acid monohydrate, 0.05 mM Na 2 E D T A and 0.4 m M octylsodium sulfate dissolved lved in distilled deionized water. This stock solution was tightly covered and kept at 4°C and an aliquot was taken as needed (usually within the same week). The workin ,orking mobile phase was prepared daily by filtering n, then adding 100 ml (10O7o) of acetonitrile ( H P L C 900 ml from the stock solution. grade) and 0.9 ml (0.09°70) o ff diethylamine. The entire mobile phase was slowly stirred and the p H was adjusted ~d to 2.55 _+ 0.01 using solid sodium hydroxide then
triction on Monoamine Levels date Nucleus)
5
6 .
•
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.
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Ma~e, Fle~ Male, ad 1 Female, F Female, ;!
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5HT
DA
5-HIAA
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Etlect of Caloric Restriction on Monoamine Levels (Hypothalamus) 5
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33
DOPAC
s 45
5-HT
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Male, Restricted M a l e , adlibitum
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Female,Restricted Female, adtibitum
5 HIAA
',d under vacuum for 20--30 min. The mobile phase was recycled overnight degassed for 16--20 -20 h in order to stabilize the analytical column and to obtain a stable baseline. Flow ow rate was 0.8 m l / m i n throughout the procedure. The T electric output of the detectorr, representing the peaks of different compounds, was quantitated using a n t e ~ r a t n r ((Hewlett-Packard l - l e w l e t t - P a c,'kard, k a r d _ Avondale, A v n n d a l e . PA). PAL 3380 A iintegrator ished by obtaining the percent (ratio) of peak areas of A standard curve was established 5 concentrations of each c o mLpOUnd (313--2500 pg//al) to that of D H B A . Tissue sample quantitation was obtained ned by comparing the ratio to the standard curve values. The final concentration off monoamines mono~ noamines and their metabolites was expressed on weight (g). Student's t-test was used for significant the basis of the original tissue differences between ad lib. andJ restricted groups. Statistical analysis was conducted separately for each sex. RESULTS
The final body weight at thee time of sacrifice was 249 ___ 22 and 459 ___ 23 g for the male restricted and ad lib. animals, respectively and 167.0 + 21 and 322.5 _+ 54.0 g for the female restricted and ad lib. rats, respectively.
3mine Levels
Male, Restricted Male, ad libitum ~
Female, Restricted
i
Femalead libitum
I.k.I I.L NE
DA
1- D
DOPAC
5-HT
5-HIAA
Effect of Caloric Restriction on Monoamine Levels Le~ (Nucleus Accumbens)
2000
9 10
10 !!~
• [] [] []
1500
Male,R Male,at Female, Female,
t--
1000 910 8
t..,
-
(9
l k!
9 ,-, 10
~> 500 ~
5533
l~I~
0 5-HT
5-HIAA
DA
DOPAC
HVA
Fig. 1. The te conc~ concentration of monoamines and their metabolites in the caudate caud~ nucleus (Fig. l-A), hypothalamus (Fig. I-B), olfactory bulb (Fig. l-C) and nucleus accumbens (Fig. l-D) 1 of 22.25-month-old male le Fischer 344 rats. Animals were maintained on an ad lib. or restricted r diet for 18 months and female :rifice. Each value is the mean _+ S.E.M. Number above each bbar represents the number of before sacrifice. rats/group. Significant differences between ~¢een restricted and adlib, in both sexes are denoted by * P < 0.05, **P< 0.01.
The effects of long-term caloric oric restriction on the concentration of monoamines and their metabolites in the CN N o f male and female Fischer 344 rats are shown in Fig. 1-A. NE, DA, 5-HT andI H V A were significantly reduced in the calorically restricted groups. NE concentration 'ation in this region was reduced by 75070 (male) and 90°70 (female) in the caloric restricted ricted animals as compared to the ad lib. control rats of the same sex. D A and 5-HT IT concentrations were reduced by 56°7o and 62070, respectively, in the CN o f male role restricted rats. The concentrations of various neurotransmitters in the H Y P O) are presented in Fig. 1-B. In the male rats, only 5H T was significantly reduced (by 36070 by 36070) 5070) in the caloric restricted animals, when compared to the ad lib. rats. In contra~ ntrast, the female rats of the caloric restricted group
(35%), DA (33%) D O P A C (23% I to ad lib. control rats. ~oth sexes of the caloric restricted J 42% (female) as compared to the ificant sex difference in the DA females of both groups had lower 'actory bulb of the male resticted r trois. There was a significant incre ale restricted animals as compared s or to the Iemale of both experimental ~erimental groups. effect of caloric restriction on monoamine concentrations concenm in in Fig. 1-D. The concentrations of 5-HT and 5-HIAA 5-HI• in thL, restricted rats were significantly reduced by 37% an and 55%, r 'ed to the ad lib. animals.
%) fifals ~C )ns
ifi7b. re{he as
~ION
preliminary study suggests that long-term caloric restrictio tly the content of various neurotransmitter in several brain re ale female Fischer 344 rats. In all brain regions studied, stuc diet on .~d to decrease the concentration of some of the monoar leir lites with a single exception of 5 - H I A A in the OB of the male restricted rats where the level was greatly increased. content in the Ther~'e are many reports indicating that diet can alter monoamine mo tryptophan availability is the or the serotonergic system, it is well known that trypt CNS. For in brain 5-HT levels rate-limitin fiting step in 5-HT synthesis. The substantial reduction reducti in caloric ric restricted rats found in this study could be a consequence c of reduced tryptophan available for dietary tryptophan, which in turn affected the amount of t~ brain uptake. On the other hand, some other types of diet di~ restriction have been reportedd to increase 5-HT concentration in the brains of undernourished t animals [13--17], perhaps due to reduced tryptophan competitive binding by serum Lncement nent of tryptophan uptake by the CNS [16--18]. albumins and subsequent enhancemen The present study showed that the decrease in the 5-HT level in both the HYPO and tease in the level of its metabolite 5-HIAA, suggesting NA was accompanied by a decrease a reduction in 5-HT synthesis. On the other hand, elevated 5-HIAA concentration in a t suggests that 5-HT is metabolized more rapidly in the OB of the male restricted rat this region, resulting in deple.~tion of 5-HT. These findings suggest that dietary restriction has complex effects on CNS 5-HT contents, effects which are regionally nd on the precise nature, onset and duration of the specific, and doubtlessly depend nutritional deprivation employed. Similarly, dietary restrictiona may well reduce catecholcomine synthesis. Tyrosine hydroxylase is the rate-limitingg enzyme in the biosynthetic pathway which converts
hat mation o f NE. Recently, it has bc lch osine hydroxylase activity in rat b ith ing effect upon neurotransmitter : ely n the sequence, such as NE being Lse in this study, where NE levels ted ntial effect is in agreement with a~ ort H Y P O of food-deprived rats [20] duced alterations in CNS monoamJ [ter mic 5-HT, NE and DA are knox~ ice y function and hence the entire hormonal mileau [21 21--24]. T1 Lin alamic monoamines found in this study could thus have a pn act he endocrine system. It has recently been reporte ~orted that c ary ion increases basal glucocorticoid levels [25], a findin~ g replicat ~o'unpublished observation). Changes in corticosterom 9ne levels, i: for ime or over the life span, could be expected to have a substant na nge of biological variables, perhaps including tg the aging agir proces~ tmmary, the present study suggests that long-term caloric cal restr ter rochemical balance in the brain. This effect occurred despite t of ogical mechanisms that protect the brain against shifts shi in the or punt of important nutrients. Conclusions from these th preli 1Its be made very cautiously until further evidence is ob! obtained on md n of caloric or diet restriction on other factors that regulate normal neurotransmitters [tters levels. These including peripheral and central concentrations c of amino acids such uch as tyrosine and tryptophan, changes in the Va~ of the rate-limiting enzymes:s of monoamines biosynthesis, the activity of metabolizing metab, enzymes and the turnover ~'r rate of these monoamines under such experimenta ~erimental conditions. It will also be necessa ;ssary to clearly distinguish between the effects of short-term (acute) and long-term rm (chronic) food deprivation on CNS monoamines. ACKNOWLEDGMENTS
The authors express their a ppreciation to Miss Kellye Walker and Ms. Barbara Jacks for preparing this manuscrt ascript. This work was supported by the Oak Ridge Associated Universities P r o g r atm m at the National Center for Toxicological Research (NCTR). REFERENCES 1 2 3
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