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Oxytocin concentration changes in different rat brain areas but not in plasma during aging
Neurobiologvo['Aging,Vol. 13, pp. 783-786, 1992
0197-4580/92 $5.00 + .00 Copyright© 1992PergamonPress Ltd.
Printed in the USA.All rightsreserved.
Oxytocin Concentration Changes in Different Rat Brain Areas But Not in Plasma During Aging M A R I A R O S A R I A MELIS, R O B E R T O S T A N C A M P I A N O , W A L T E R F R A T T A AND ANTONIO ARGIOLAS 1
Bernard B. Brodie Department of Neuroscience, University of Cagliari, Via Porcell 4, 09124 Cagliari, Italy R e c e i v e d 14 F e b r u a r y 1992; Accepted 23 J u n e 1992 MELIS, M. R., R. STANCAMPIANO, W. FRATTA AND A. ARGIOLAS. Ox),tocinconcentration changes in different rat brain areas but not in plasma during aging. NEUROBIOL AGING 13(6)783-786, 1992.--The concentration ofoxytocin was measured by radioimmunoassay in different brain areas, hypophysis, and plasma of male Wistar Kyoto rats during aging. Although no difference in the concentration ofoxytocin in any of the above tissues among 2- and 6-month-old rats was found, in 12-month-old rats a 21% decrease was observed in both septum and hippocampus, but not in the hypothalamus, hypophysis, and plasma, when compared to values of 2- and 6-month-old rats. In 18-month-old rats, the decrease ofseptal and hippocampal oxytocin content was higher than that found in 12-month-old rats, but no change was found in the hypothalamus, neurohypophysis, and plasma. In 24-month-old rats, oxytocin content was similar to that found in 18-month-old rats in all tissues analyzed. The results suggest that aging induces an impairment of oxytocinergic transmission in the central nervous system but not in the neurohypophyseal system. Oxytocin
Aging
Brain areas
Plasma
Rat
OXYTOCIN, the nonapeptide of the neurohypophysis wellknown for its hormonal role in parturition and lactation, is present not only in the hypothalamic neurohypophyseal system but also in other areas of the central nervous system (CNS). Accordingly, neuroanatomical and immunocytochemical studies have shown that the paraventricular nucleus of the hypothalamus (PVN) contains not only the cell bodies of magnocellular neurons projecting to the neurohypophysis, from which oxytocin is released into the circulation, but also parvocellular neurons that send their projections to several extrahypothalamic brain areas, such as amygdala, septum, hippocampus, olfactory tubercle, pons, medulla, and spinal cord (6,25). These oxytocinergic neuronal pathways are thought to be involved in several central functions, such as memory and learning, maternal behavior, sexual behavior, drug tolerance and dependence, feeding, cardiovascular and autonomic regulation [for a review see (3,22)]. Because an impairment of many of these functions is observed during aging, and this may be related to a modification of central oxytocinergic transmission, we measured the concentration of oxytocin in different brain areas of rats of different months of age by radioimmunoassay (RIA).
METHOD Male Wistar Kyoto rats of 2, 6, 12, 18, and 24 months of age were provided by Charles River (Como, Italy). The rats were housed in groups of 4-6 at 22* + I°C with standard laboratory food and tap water ad lib.
Oxytocin Extraction Oxytocin extraction from brain tissues and plasma was performed as previously described in detail (2,20). Briefly, rats were sacrificed by decapitation and the brains were quickly removed. The hypophysis, hypothalamus, hippocampus, and septum were dissected out on a cold glass slide, rapidly transferred into 16 × 125 m m polyethylene tubes containing 2 ml of ice-cold 2M acetic acid. Tissues were boiled for 10 min in a water bath and homogenized. An aliquot was removed to determine protein content (19). After centrifugation at × 28,000 g for 20 min, the supernatant was concentrated under vacuum with a Speed Vac (Savant). The samples were then dissolved in RIA buffer and assayed for oxytocin as described below. Under these conditions, the recovery of ['25]I-oxytocin added to the homogenization medium at the beginning of the procedure
~To whom requests for reprints should be addressed. 783
784
MEI.IS E l Al,.
was 82%-85%. Blood was collected from the trunk alter decapitation in 16 × 125 m m polyethylene tubes containing EDTA as anticoagulant and 10 IU Trasylol (Sigma, S. Louis, MO). After centrifugation at × 1000 g for 15 min, oxytocin was extracted from the supernatant and assayed by RIA.
O.vytocin RIA Oxytocin antibody was raised in New Zealand rabbits by repeated injections of a mixture of synthetic oxytocin coupled to bovine serum albumine and Freund's complete adjuvant supplemented with 5 mg/kg killed tubercolosis mycobacterium (Difco). Animals were inoculated once every 3 weeks for 9 weeks followed by bimonthly booster injections. Antibody was collected by heart puncture 6 months after initial injections. The antibody showed 0.01% crossreactivity with argS-vasopressin and vasotocin. Oxytocin content was determined by incubating the antibody (final dilution 1 : 12,000) with samples dissolved in RIA buffer (0.05 M sodium phosphate buffer, pH 7.4, containing 0.125% bovine serum albumine, 0.05% L-cystine and 0.1% sodium azide) and 6000 cpm of [t25-1]oxytocin. Free and bound [~25-I]oxytocin were separed by means ofdextrane-coated charcoal. [~25-I]oxytocin was prepared by the cioramine T method and purified by gel filtration on a Sephadex G25. Oxytocin (Peninsula Laboratories, CA) was used as the standard. Under our conditions, the linear range of the assay was 0.5-50 pg/tube, the inter- and intra-assay variations 12% and 18%, respectively. Under the conditions of the assay, serial dilutions of plasma, hypothalamic, hippocampal, and septal extracts gave displacements of [J25-I] oxytocin parallel to that of authentic oxytocin.
Statistics Statistical evaluation was performed by one way ANOVA, followed by Duncan's multiple range test. RESULTS Table 1 shows the concentration of immunoreactive oxytocin in the hypothalamus, septum, hippocampus, hypophysis, and plasma of 2-, 6-, 12-, 18-, and 24-month-old rats. No significant difference was found between 2- and 6-month-old rats in any of the tissues analyzed. A small but significant 21% decrease in oxytocin content was found in both septum and hippocampus in i 2-month-old rats, when compared to 2- or 6-
month-old rats. Such a decrease was more marked in the septum (30%) and hippocampus (40%) of 18-month-old rats, which also showed a 14% decrease in hypothalamic oxytocin. A slight but not significant decrease in oxytocin content was tbund in the hypophysis and in plasma. No turther decrease in oxytocin concentration was found in the above brain areas, hypophysis, and plasma of 24-month-old rats DISCUSSION It is widely accepted that the function of central neurotransmitter/neuromodulator systems changes during aging and that these changes are involved in some of the alterations observed during aging, i.e., impaired memory and learning processes, motor and behavioral disturbances and so on. The molecular basis of the above functional changes may be due to neuronal losses, which lead to a decreased content of neurotransmitters, changes in the mechanisms regulating neurotransmitter turnover (synthesis, release, and inactivation), in number and/or affinity of receptors for the neurotransmitter, or alterations in the transduction mechanisms (second messenger systems). Although numerous reports have shown alterations of cholinergic (12), dopaminergic (13,26), noradrenergic (9), serotoninergic (26), and gabaergic ( 1) systems during aging, very little is known about the alteration of central neuropeptidergic systems during aging. In particular, it has been shown that substance P, somatostatin, neurotensin, vasoactive intestinal polypeptide, and neuropeptide Y decrease in some brain areas of aged rats (5,15) and that the number of opiate receptors decreases during aging (7). As far as oxytocin and vasopressin are concerned, the studies available on the function of the neurohypophyseal systems during aging provided conflicting results, suggesting either a decrease ( 11,16,17,24,29,30) or an increase (10) in the activity of the vasopressinergic system. Other studies have shown that vasopressin content and projections decrease during aging in different brain areas including the hypothalamus in rats (8,23), and that this effect is reversed by testosterone (14). The above studies showed also a slight but significant decrease in oxytocin projections to the locus coeruleus, that was not reversed by testosterone (14). The above report apart, very little is available on central oxytocinergic systems in aged rats. To our knowledge, this is the first report on the effect of aging on oxytocin content in different brain areas, hypophysis, and plasma of male rats. The present results show that the concentration of oxytocin decreases during aging in
TABLE l IMMUNOREACTIVEOXYTOCINCONCENTRATION IN DIFFERENTBRAINAREAS, HYPOPHYSISAND PLASMAOF MALEWISTARKYOTO RATS DURINGAGING Brain Areas Hypothalamus Age(Months) 2 6 12 18 24
N 18 22 20 22 9
ng/mg prot 2.21 2.20 2.16 t.96 1.95
_+ 0.32 + 0.23 + 0122 _+ 0.26 + 0.32
Septum
Hippocampus Hypophysis
pg/mg prot
pg/mgprot
~g/mgprot
132 125 105 95 93
9.7 9.2 7.7 5.0 5.6
2.1 2.0 2.1 1.8 1.8
+ 21 + 26 + 12" _+ 11" + 13"
_+ 0.9 _+ 1.0 _+ 0.9* + 0.5* _+ 0.6*
+ 0.2 _+ 0.3 _+ 0.5 _ 0.9 _+ 1.1
Plasma pg/ml 2.3 _+ 0.4 2.7 _+ 0.3 2.8 + 0.4 2.0 _+ 0.8 1.9 + 0.8
Oxytocin was determined by RIA. Values are mean ± SEM of the number of the rats reported here. *P
BRAIN OXYTOCIN AND AGING
785
the septum and, to a major extent, in the hippocampus but not in the hypothalamus, hypophysis, or plasma. Such a decrease in oxytocin content might be due to a loss of oxytocinergic neurons or to a decreased synthesis of the peptide at hypothalamic level, or to an increased a m o u n t of non-neuronal proteins, secondary to neuronal losses during aging. The first possibility is the most likely, being in agreement with the above recalled small but significant decrease in central oxytocinergic projections found in aged rats (14). The finding suggests an impaired central oxytocinergic function in these brain structures. In contrast, the neurohypophyseal oxytocinergic activity does not appear to be modified during aging, as indicated by the absence of changes in oxytocin content in the hypothalamus, hypophysis, and plasma of aged rats. The finding is in agreement with previous studies showing that the size and number of oxytocinergic cell bodies in the hypothalamic supraoptic and paraventricular nucleus of post mortem human brain did not change during aging or senile dementia (10,28). In this respect, the neurohypophyseal oxytocinergic system seems different from that vasopressinergic during aging. Indeed, as recalled above, the activity of such system as well as hypothalamic vasopressin content decrease during aging (8,11,16,24,29,30). In view of the well-established role of the septum and hip-
pocampus in cognitive processes, emotional, and reproductive behavior, it is tempting to speculate that central oxytocin and vasopressin reduction is involved in the impairment of the above functions observed during aging. In agreement with this hypothesis, (a) septum and hippocampus are thought to mediate the effects of neurohypophyseal peptides on memory and learning processes [see (3) and enclosed references], (b) oxytocin and, to a lesser extent, vasopressin excite hippocampal neurons by acting on uterine-type oxytocinergic receptors (21 ), (c) oxytocin stimulates sexual behavior of aging male rats (4) and (d) it has been reported that vasopressin improves performance in tests involving attention and memory in 50-60-yearold men (18,27). In conclusion, our results show that in male rats oxytocin content decreases in brain more than in the neurohypophyseal system during aging. ACKNOWLEDGEMENTS The research activities leading to this work were supported by the National Program of Pharmacological Research (Rif. 1139/191078603), implemented by the Italian Ministry of University and Scientific and Technological Research.
REFERENCES 1. Allen, S. J.; Benton, J. S.; Goodhardt, M. J.; Haan, E. A.; Sims, N. M.; Smith, C. C. T.; Spillane, J. A.; Bowen, D. M.; Davison, A. N. Biochemical evidence of selective nerve cell changes in normal ageing human and rat brain. J. Neurochem. 41:256-265; 1983. 2. Amico, A. J.; Ervin, M. G.: Leake, R. D.; Fisher, D. A.; Finn, F. M.; Robinson, A. G. A novel oxytocin-like and vasotocin-like in human plasma after administration of estrogen. J. Clin. Endocrinol. Metab. 60:5-13; 1985. 3. Argiolas, A.; Gessa, G. L. Central functionsofoxytocin. Neurosci. Biobehav. Rev. 15:217-231; 1991. 4. Arletti, R.; Benelli, A.; Bertolini, A. Sexual behavior of aging male rats is stimulated by oxytocin. Eur. J. Pharmacol. 179:377-381: 1990. 5. Buck, S. H.; Deshmukh, P. P.; Burks, T. F.; Yamamura, H. I. A survey of substance P, somatostatin and neurotensin levels in aging in the rat and human central nervous system. Neurobiol. Aging 2:257-264:1981. 6. Bujis, R. M. Intra- and extrahypothalamic vasopressin and oxytoein pathways in the rat. Cell Tissue Res. 192:423-435; 1978. 7. Chee, C. A.; Roozendaal, B.; Swaab, D. F.; Goudsmit, E.; Mirmiran, M. Vasoactive intestinal polypeptide neuron changes in the senile rat suprachiasmatic nucleus. Neurobiol. Aging 9:307312; 1988. 8. Dorsa, D. M.; Bottemiller, L. Age-related changes of vasopressin content of microdisseeted areas of the rat brain. Brain Res. 242:151-155; 1982. 9. Eriksdotter-Nilson, M.; Gerhardt, G.; Seiger, A.; Hoffer, B.; Granholm, A. C. Multiple changes in noradrenergic mechanisms in the coeruleo-hippocampal pathway during aging: structural and functional correlates in intraocular double grafts. Neurobiol. Aging 10:117-124; 1989. 10. Fliers, E.; Swaab, D. F.; Pool, Chr. W.; Verwer, R. W. H. The vasopressin and oxytocin neurons in the human supraoptic and paraventricular nucleus; changes with aging and in senile dementia. Brain Res. 342:45-53; 1985. 11. Friedman, S. M.; Sreter, F. A.; Friedman, C. L. The effect ofvasopressin and aldosterone on the distribution of water, sodium and potassium and on work performance in old rats. Gerontologia 7:65-76; 1963. 12. Fu Wu, C.; Bartorelli, R.; Sacconi, M.; Pepeu, G.; Consolo, S. Decrease of brain acetylcholine release in aging freely-movingrats detected by microdialysis. Neurobiol. Aging 9:357-361; 1988. 13. Giorgi, O.; Porceddu, M. L.; De Montis, G.; Toffano, G.; Biggio,
G. D-1 dopamine receptors in the brain and retina of aged rats:
14. 15. 16.
17.
18. 19. 20.
21. 22. 23. 24. 25. 26.
effect of chronic blockade by SCH 23390. In: Pepeu, G.; Tomlinson, B.; Wischik, C. M., eds. New trends in aging research. Padova: liviana Press; 1988:37-46. Goudsmit, E.; Fliers, E.; Swaab, D. F. Testosterone supplementation restores vasopressin innervation in the senescent rat brain. Brain Res. 473:306-313; 1988. Higuchi, H.; Hsiu-Ying, T.; Yang, T. Age-related re-directional changes in neuropeptide Y peptides in rat adrenal glands, brain and blood. J. Neurochem. 50:1879-1885: 1988. Kawashima, S.; Kawamoto, K. Age-related changes in plasticity of vasopressin- and oxytocin-immunoreactive neurons in rats and mice. In: Pepeu, G.; Tomlinson, B.; Wischik, C. M,, eds. New trends in aging research. Padova: Liviana Press; 1988:67-74. Legros, J. J. The radioimmunoassay of human neurohypophysins: Contributions to the understanding of the physiopathology of neurohypophyseal function. Ann. N.Y. Acad. Sci. 248:281303; 1975. Legros, J. J.; Gilot, P.; Seron, S.: Claesse, J.; Adam, A.; Moeglen, J. M.; Audibert, A.; Berchier, P. Influence ofvasopressin on learning and memory. Lancet 1:41-42: 1978. Lowry, O. H.: Rosebrough, N. J.; Farr, A. L.: Randall, R. H. Protein measurement with the pholin phenol reagent. J. Biol. Chem. 193:265-275; 1951. Melis, M. R.; Argiolas, A.; Stancampiano, R.; Gessa, G. h Effect of apomorphine on oxytocin concentrations in different brain areas and plasma of male rats. Eur. J. Pharmacol. 182:101-107: 1990. Muhlethaler, M.; Sawyer, W. H.; Manning, M. M.; Dreifuss, J. J. Characterization of a uterine-type oxytocin receptor in the rat hippocampus. Proc. Natl. Acad. Sci. U.S.A. 80:6713-6717; 1983. Richard, P.; Moos, F.; Freund-Mercier, M. J. Central effects of oxytocin. Physiol. Rev. 71:331-370; 1990. Roozendaal, B.; van Gool, W. A.; Swaab, D. F.; Hoogendijk, J. E.; Mirmiran, M. Changes in vasopressin cells of the rat suprachiasmatic nucleus with aging. Brain Res. 409:259-264; 1987. Sladek, C. D.; McNeill, T. H.; Gregg, C. M.; Grail, M. L.; Baggs, R. B. Vasopressin and renin response to dehydration in aged rats. Neurobiol. Aging 2:293-297; 1981. Sofroniew, M. W. Vasopressin and oxytocin in mammalian brain and spinal cord. Trends Neurosci. 6:467-472: 1983. Steinbuseh, H. W. M.; Wolters, J. G.; Van Luijtelaar, M. G. P. A.; Tonnaer, J. A. D. M. The serotoninergic and dopaminergic sys-
786 tems in the aged rat brain as studied by immunohistochemistD with antibodies to serotonin and dopamine. In: Pepeu, G.; Tomlinson, B.; Wischik, C. M., eds. New trends in aging research. Padova: Liviana Press; 1988:193-203, 27. Weingartner, H.; Kaye, W.; Gold, P.; Smallberg, S.; Peterson, R.; Gillin, J. C.; Ebert, M. Vasopressin treatment of cognitive dysfunction in progressive dementia. Life Sci. 29:2721-2726; 1981. 28. Wierda, M.; Goudsmit, E.; Van Der Woude, P. F.; Purba, J. S.: Hofman, M. A.: Bogte, H.; Swaab, D. F. Oxytocin cell number in
MELIS E t ,\I.. the human paraventricular nucleus remains constant with aging and in Alzheimer's disease. Neurobiol. Aging 12:511-516; 199t. 29. Zbuzek, V. K.; Wu, W.-H. Age-related vasopressin changes in rat plasma and the hypothalamo-hypophyseal system. Exp. Gerontol. 17:133-138; 1982. 30. Zbuzek, V. K.; Zbuzek, V.; Wu, W.-H. Age-related diflerences in the incorporation of 3H-arginine into vasopressin in Fisher 344 rats. Exp. Gerontol. 22:113-126~ 1987.
0197-4580/92 $5.00 + .00 Copyright© 1992PergamonPress Ltd.
Printed in the USA.All rightsreserved.
Oxytocin Concentration Changes in Different Rat Brain Areas But Not in Plasma During Aging M A R I A R O S A R I A MELIS, R O B E R T O S T A N C A M P I A N O , W A L T E R F R A T T A AND ANTONIO ARGIOLAS 1
Bernard B. Brodie Department of Neuroscience, University of Cagliari, Via Porcell 4, 09124 Cagliari, Italy R e c e i v e d 14 F e b r u a r y 1992; Accepted 23 J u n e 1992 MELIS, M. R., R. STANCAMPIANO, W. FRATTA AND A. ARGIOLAS. Ox),tocinconcentration changes in different rat brain areas but not in plasma during aging. NEUROBIOL AGING 13(6)783-786, 1992.--The concentration ofoxytocin was measured by radioimmunoassay in different brain areas, hypophysis, and plasma of male Wistar Kyoto rats during aging. Although no difference in the concentration ofoxytocin in any of the above tissues among 2- and 6-month-old rats was found, in 12-month-old rats a 21% decrease was observed in both septum and hippocampus, but not in the hypothalamus, hypophysis, and plasma, when compared to values of 2- and 6-month-old rats. In 18-month-old rats, the decrease ofseptal and hippocampal oxytocin content was higher than that found in 12-month-old rats, but no change was found in the hypothalamus, neurohypophysis, and plasma. In 24-month-old rats, oxytocin content was similar to that found in 18-month-old rats in all tissues analyzed. The results suggest that aging induces an impairment of oxytocinergic transmission in the central nervous system but not in the neurohypophyseal system. Oxytocin
Aging
Brain areas
Plasma
Rat
OXYTOCIN, the nonapeptide of the neurohypophysis wellknown for its hormonal role in parturition and lactation, is present not only in the hypothalamic neurohypophyseal system but also in other areas of the central nervous system (CNS). Accordingly, neuroanatomical and immunocytochemical studies have shown that the paraventricular nucleus of the hypothalamus (PVN) contains not only the cell bodies of magnocellular neurons projecting to the neurohypophysis, from which oxytocin is released into the circulation, but also parvocellular neurons that send their projections to several extrahypothalamic brain areas, such as amygdala, septum, hippocampus, olfactory tubercle, pons, medulla, and spinal cord (6,25). These oxytocinergic neuronal pathways are thought to be involved in several central functions, such as memory and learning, maternal behavior, sexual behavior, drug tolerance and dependence, feeding, cardiovascular and autonomic regulation [for a review see (3,22)]. Because an impairment of many of these functions is observed during aging, and this may be related to a modification of central oxytocinergic transmission, we measured the concentration of oxytocin in different brain areas of rats of different months of age by radioimmunoassay (RIA).
METHOD Male Wistar Kyoto rats of 2, 6, 12, 18, and 24 months of age were provided by Charles River (Como, Italy). The rats were housed in groups of 4-6 at 22* + I°C with standard laboratory food and tap water ad lib.
Oxytocin Extraction Oxytocin extraction from brain tissues and plasma was performed as previously described in detail (2,20). Briefly, rats were sacrificed by decapitation and the brains were quickly removed. The hypophysis, hypothalamus, hippocampus, and septum were dissected out on a cold glass slide, rapidly transferred into 16 × 125 m m polyethylene tubes containing 2 ml of ice-cold 2M acetic acid. Tissues were boiled for 10 min in a water bath and homogenized. An aliquot was removed to determine protein content (19). After centrifugation at × 28,000 g for 20 min, the supernatant was concentrated under vacuum with a Speed Vac (Savant). The samples were then dissolved in RIA buffer and assayed for oxytocin as described below. Under these conditions, the recovery of ['25]I-oxytocin added to the homogenization medium at the beginning of the procedure
~To whom requests for reprints should be addressed. 783
784
MEI.IS E l Al,.
was 82%-85%. Blood was collected from the trunk alter decapitation in 16 × 125 m m polyethylene tubes containing EDTA as anticoagulant and 10 IU Trasylol (Sigma, S. Louis, MO). After centrifugation at × 1000 g for 15 min, oxytocin was extracted from the supernatant and assayed by RIA.
O.vytocin RIA Oxytocin antibody was raised in New Zealand rabbits by repeated injections of a mixture of synthetic oxytocin coupled to bovine serum albumine and Freund's complete adjuvant supplemented with 5 mg/kg killed tubercolosis mycobacterium (Difco). Animals were inoculated once every 3 weeks for 9 weeks followed by bimonthly booster injections. Antibody was collected by heart puncture 6 months after initial injections. The antibody showed 0.01% crossreactivity with argS-vasopressin and vasotocin. Oxytocin content was determined by incubating the antibody (final dilution 1 : 12,000) with samples dissolved in RIA buffer (0.05 M sodium phosphate buffer, pH 7.4, containing 0.125% bovine serum albumine, 0.05% L-cystine and 0.1% sodium azide) and 6000 cpm of [t25-1]oxytocin. Free and bound [~25-I]oxytocin were separed by means ofdextrane-coated charcoal. [~25-I]oxytocin was prepared by the cioramine T method and purified by gel filtration on a Sephadex G25. Oxytocin (Peninsula Laboratories, CA) was used as the standard. Under our conditions, the linear range of the assay was 0.5-50 pg/tube, the inter- and intra-assay variations 12% and 18%, respectively. Under the conditions of the assay, serial dilutions of plasma, hypothalamic, hippocampal, and septal extracts gave displacements of [J25-I] oxytocin parallel to that of authentic oxytocin.
Statistics Statistical evaluation was performed by one way ANOVA, followed by Duncan's multiple range test. RESULTS Table 1 shows the concentration of immunoreactive oxytocin in the hypothalamus, septum, hippocampus, hypophysis, and plasma of 2-, 6-, 12-, 18-, and 24-month-old rats. No significant difference was found between 2- and 6-month-old rats in any of the tissues analyzed. A small but significant 21% decrease in oxytocin content was found in both septum and hippocampus in i 2-month-old rats, when compared to 2- or 6-
month-old rats. Such a decrease was more marked in the septum (30%) and hippocampus (40%) of 18-month-old rats, which also showed a 14% decrease in hypothalamic oxytocin. A slight but not significant decrease in oxytocin content was tbund in the hypophysis and in plasma. No turther decrease in oxytocin concentration was found in the above brain areas, hypophysis, and plasma of 24-month-old rats DISCUSSION It is widely accepted that the function of central neurotransmitter/neuromodulator systems changes during aging and that these changes are involved in some of the alterations observed during aging, i.e., impaired memory and learning processes, motor and behavioral disturbances and so on. The molecular basis of the above functional changes may be due to neuronal losses, which lead to a decreased content of neurotransmitters, changes in the mechanisms regulating neurotransmitter turnover (synthesis, release, and inactivation), in number and/or affinity of receptors for the neurotransmitter, or alterations in the transduction mechanisms (second messenger systems). Although numerous reports have shown alterations of cholinergic (12), dopaminergic (13,26), noradrenergic (9), serotoninergic (26), and gabaergic ( 1) systems during aging, very little is known about the alteration of central neuropeptidergic systems during aging. In particular, it has been shown that substance P, somatostatin, neurotensin, vasoactive intestinal polypeptide, and neuropeptide Y decrease in some brain areas of aged rats (5,15) and that the number of opiate receptors decreases during aging (7). As far as oxytocin and vasopressin are concerned, the studies available on the function of the neurohypophyseal systems during aging provided conflicting results, suggesting either a decrease ( 11,16,17,24,29,30) or an increase (10) in the activity of the vasopressinergic system. Other studies have shown that vasopressin content and projections decrease during aging in different brain areas including the hypothalamus in rats (8,23), and that this effect is reversed by testosterone (14). The above studies showed also a slight but significant decrease in oxytocin projections to the locus coeruleus, that was not reversed by testosterone (14). The above report apart, very little is available on central oxytocinergic systems in aged rats. To our knowledge, this is the first report on the effect of aging on oxytocin content in different brain areas, hypophysis, and plasma of male rats. The present results show that the concentration of oxytocin decreases during aging in
TABLE l IMMUNOREACTIVEOXYTOCINCONCENTRATION IN DIFFERENTBRAINAREAS, HYPOPHYSISAND PLASMAOF MALEWISTARKYOTO RATS DURINGAGING Brain Areas Hypothalamus Age(Months) 2 6 12 18 24
N 18 22 20 22 9
ng/mg prot 2.21 2.20 2.16 t.96 1.95
_+ 0.32 + 0.23 + 0122 _+ 0.26 + 0.32
Septum
Hippocampus Hypophysis
pg/mg prot
pg/mgprot
~g/mgprot
132 125 105 95 93
9.7 9.2 7.7 5.0 5.6
2.1 2.0 2.1 1.8 1.8
+ 21 + 26 + 12" _+ 11" + 13"
_+ 0.9 _+ 1.0 _+ 0.9* + 0.5* _+ 0.6*
+ 0.2 _+ 0.3 _+ 0.5 _ 0.9 _+ 1.1
Plasma pg/ml 2.3 _+ 0.4 2.7 _+ 0.3 2.8 + 0.4 2.0 _+ 0.8 1.9 + 0.8
Oxytocin was determined by RIA. Values are mean ± SEM of the number of the rats reported here. *P
BRAIN OXYTOCIN AND AGING
785
the septum and, to a major extent, in the hippocampus but not in the hypothalamus, hypophysis, or plasma. Such a decrease in oxytocin content might be due to a loss of oxytocinergic neurons or to a decreased synthesis of the peptide at hypothalamic level, or to an increased a m o u n t of non-neuronal proteins, secondary to neuronal losses during aging. The first possibility is the most likely, being in agreement with the above recalled small but significant decrease in central oxytocinergic projections found in aged rats (14). The finding suggests an impaired central oxytocinergic function in these brain structures. In contrast, the neurohypophyseal oxytocinergic activity does not appear to be modified during aging, as indicated by the absence of changes in oxytocin content in the hypothalamus, hypophysis, and plasma of aged rats. The finding is in agreement with previous studies showing that the size and number of oxytocinergic cell bodies in the hypothalamic supraoptic and paraventricular nucleus of post mortem human brain did not change during aging or senile dementia (10,28). In this respect, the neurohypophyseal oxytocinergic system seems different from that vasopressinergic during aging. Indeed, as recalled above, the activity of such system as well as hypothalamic vasopressin content decrease during aging (8,11,16,24,29,30). In view of the well-established role of the septum and hip-
pocampus in cognitive processes, emotional, and reproductive behavior, it is tempting to speculate that central oxytocin and vasopressin reduction is involved in the impairment of the above functions observed during aging. In agreement with this hypothesis, (a) septum and hippocampus are thought to mediate the effects of neurohypophyseal peptides on memory and learning processes [see (3) and enclosed references], (b) oxytocin and, to a lesser extent, vasopressin excite hippocampal neurons by acting on uterine-type oxytocinergic receptors (21 ), (c) oxytocin stimulates sexual behavior of aging male rats (4) and (d) it has been reported that vasopressin improves performance in tests involving attention and memory in 50-60-yearold men (18,27). In conclusion, our results show that in male rats oxytocin content decreases in brain more than in the neurohypophyseal system during aging. ACKNOWLEDGEMENTS The research activities leading to this work were supported by the National Program of Pharmacological Research (Rif. 1139/191078603), implemented by the Italian Ministry of University and Scientific and Technological Research.
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