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Cyclic AMP and cyclic GMP accumulation in vitro in brain regions of young, old and aged rats
Brain Re.,earch, 139 (1978) 169-177 © Elsevier/North-Holland Biomedical Press
169
Cyclic AMP and cyclic GMP accumulation in vitro in brain regions of young, old and ,aged rats
MICHAEL J. SCHMIDT* and JOHN F. THORNBERRY Lilly Research Laboratories, Eli Lilly and Company Indianapolis, Ind. 46206 (U.S.A.)
(Accepted August 8th, 1977)
Ultrastructural changes in synaptic morphology 11 or abnormalities in neurotransmitter-receptor interaction might account for the functional defects often seen in the aged. Neurotransmitter-stimulated synthesis of cyclic nucleotides in brain slices has been widely used as a biochemical measure of receptor activity 5, and the ability of norepinephrine to stimulate adenosine 3'5'-monophosphate (cAMP) synthesis in brain tissue is age-dependent. Norepinephrine does not elevate cAMP levels in brain slices until 5-6 days after birth zs, even though adenylate cyclase is present in the rat brain at birth. A similar phenomenon was seen in the rabbit brain, but in this species a marked reduction in hormone-stimulated c A M P synthesis occurred in old animals 29. The latter studies suggested that a reduction in transmitter-stimulated c A M P accumulation might contribute to the mental dysfunction which often accompanies old age. The purpose of the present studies was to survey transmitter-receptor interactions in young, old and aged rats by monitoring norepinephrine-stimulated accumulation of cyclic nucleotides in slices of brain regions incubated in vitro. Rats older than 3 months of age were obtained from a colony of aged Wistar rats maintained by Harlan Industries, Indianapolis, Ind. Three-month-old rats came from general Harlan stock. All rats were maintained under conditions which meet or surpass AALAS standards. Rats in the colony were examined daily and any animal with demonstrable abnormalities (tumors, blindness, excessive weight loss) was eliminated. A 12-h light-dark cycle was in effect at all times. Animals were fed and watered ad libitum. The ages chosen for study were based on the suggestion of Finch s that mice at the age of average longevity are comparable to human males 70-80 years old. Twentyfour months of age represents the 50 ~ survival level in our colony and was selected as the upper end of the age scale. Three-month-old rats were selected as the youngest group since at this age rats are sexually mature and past the stage of rapid brain growth. A 12-month-old group was included to give some indication whether our
* To whom correspondence should be addressed.
170 findings were a reflection of aging (12 months vs. 24 months), maturation (3 months vs. 12 months) or an aging continuum (3 vs. 12 vs. 24 months). Male Wistar rats were killed by decapitation, the brains removed and dissected la and brain areas minced on an ice-cooled plate. Cyclic nucleotide accumulation was determined in chopped tissue slices as described previously 3a. It was necessary to preincubate some regions longer than others in order to achieve stable basal levels of cAMP: i.e. brain stem, hypothalamus and hippocampus - - 30 rain; cerebral cortex --- 60 min; c e r e b e l l u m - 120 rain. Purification of cyclic nucleotides by sequential alumina and Dowex 1 chromatography, and separation of cAMP and guanosine 3',5'monophosphate (cGMP) by clution with 0.05 N HC1 and 0.5 N HC1, respectively, has been described 31. Recovery of cAMP was approximately 60 ~ and c G M P approximately 40 ~ using this method. Assay values of cyclic nucleotide concentrations were corrected for this loss during purification. Final results are expressed as pmoles of cyclic nucleotide per mg protein per incubation time. Adenylate cyclase activity was determined by measuring the conversion of [32p]aATP to [3ZP]cAMP and separating and purifying the end-product by the dual column procedure outlined above 25. Cyclic AMP concentrations from tissue slice experiments were determined using a protein binding assay 12, and c G M P levels by immunoassay (Schwartz-Mann, Orangeburg, N.Y.). Protein concentration was measured by the method of Lowry et al. 16 using bovine serum albumin as the standard. Kainic acid was obtained from Sigma Chemical (St. Louis, Mo.). The remaining chemicals and supplies were purchased from other commercial sources. Norepinephrine stimulated cAMP accumulation in all brain areas at all ages although the degree of stimulation varied across regions (Fig. 1). The cerebellum was the only area in which a significant age-related difference in cAMP accumulation was observed in tissue slices. The norepinephrine-elicited elevation in cAMP was 44 lower in the cerebellum of 12-month-old rats and 57 ~ lower in 24-month-old rats in comparison to 3-month-old animals. Resting levels of cAMP were also lower in slices of the cerebellum from 24-month-old rats, but in all other regions basal levels of cAMP were similar across ages. Age-related declines in basal and stimulated cAMP accumulation have been observed in adipose tissue10, is, lymphocytes iv, erythrocytes 3a and the liver 2. We demonstrated that norepinephrine-stimulated cAMP accumulation was higher in whole rat brain at 15 days of age than at 25 days of age 3°, and Weiss and Strada as found similar decreases in adenylate cyclase activity in several brain regions between 15 and 60 days of age. All of these studies reveal age-related declines in hormonereceptor interaction, but the differences actually reflect maturation rather than aging for in no instance were animals used which approached the two year point which approximates the senescent condition in rats. The present findings indicate that there is not a progressive decline in the concentration of cAMP in unstimulated tissue slices from the hypothalamus, cerebral cortex, hippocampus, or brain stem as young mature Wistar rats grow into old age. Puri and Volicer 24 also found no change in cAMP levels in the striatum of aged rats. At variance with our results are the findings of Zimmerman and Berg1, a°. They
171 observed a significant reduction in cortical c A M P in Fisher 344 rats at 6 m o n t h s o f age when c o m p a r e d to 3 - m o n t h - o l d rats. H o w e v e r , again this difference seems to represent a m a t u r a t i o n a l change for there was no further decline in c A M P levels during the last 18 m o n t h s o f the animal's life when the behavioral manifestations o f aging are seen 14. Berg and Z i m m e r m a n 1 also r e p o r t e d that in older rats there was less accumulation o f c A M P in cortical tissues which had been exposed to n o r e p i n e p h r i n e in situ. O u r results are in disagreement with these findings. It is possible the strain differences (Fisher 344 vs. Wistar) account for the disparity. H o w e v e r , m e t h o d o l o g i c a l differences
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Fig. 1. Basal and norepinephrine-stimulated accumulation of cyclic AMP in slices of brain regions from young, old and aged rats. Brain regions from individual rats were minced, rinsed with KrebsRinger buffer and preincubated in 20 ml of buffer for varying times at 37 °C as indicated in Methods. Then the medium was replaced with 9.9 ml of fresh, cold medium, and 15 min later 0.1 ml of an aqueous solution of norepinephrine or water alone was added to eadh flask, such that the final concentration of norepinephrine was 10-5 M (except the cerebellum where 10-4 M norepinephrine was used). The reaction was terminated by adding perchloric acid and homogenizing the tissues at the time of maximal cAMP accumulation in each respective area: brain stem and hippocampus - - 6 rain; hypothalamus - - 10 rain; cerebellum - - 15 min; and cerebral cortex - - 20 min. Cyclic AMP was purified by sequential alumina and Dowex 1 chromatography. Values represent the mean pmoles cAMP/rag protein :~ S.E.M. Solid bars depict cyclic AMP concentrations in the absence of norepinephrine. The variation in basal levels of cAMP within groups was approximately 10 ~ .
172 3 MONTH 12 ,, 100
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KAINATE
Fig. 2. Basal and kainate-stimulated accumulation of cyclic GMP in cerebellar slices from young, old and aged rats. Cerebellar slices from individual rats were prepared and incubated as indicated in Fig. 1. After 2 h 15 min of preincubation, kainate was added to the incubation medium and the incubation continued for 15 min. Values represent the mean ± S.E.M. of 11-13 individual determinations, each of which was assayed in duplicate for cGMP concentration. Values which differ significantly from the 3-month-old rats are indicated (*); P < 0.05, Student's t-test. between our studies and those of Zimmerman and Berg might also explain the lack of agreement. For example, Berg and Zimmerman 1 estimated hormone stimulation by adding norepinephrine to cotton pledgets placed on the cortex of anesthetized rats. Dose-response relationships cannot be controlled under these conditions and previous studies have shown that anesthesia reduces cAMP levels in vivo 26. Furthermore, the rats were sacrificed by decapitation prior to tissue fixation and this produces differences in c A M P concentrations in all brain regions z~. In our experiments the basal and norepinephrine-stimulated levels of c A M P observed in cortical slices from 3month-old rats (Fig. 1) are comparable to the work of others zz. Cyclic G M P levels were determined in brain regions during control incubations. The level o f c G M P was much lower than c A M P in most all areas: cerebral cortex (0.31 ~- 0.05), hippocampus (0.45 ~: 0.06), hypothalamus (0.44 ± 0.03) and brain stem (0.42 z~ 0.08 pmoles/mg protein). Concentrations were similar at 3, 12 and 24 months of age in these areas. However, in the cerebellum the concentration of c G M P was significantly lower at 24 months of age than at 12 or 3 months (Fig. 2). When cerebellar slices were stimulated by the addition of kainic acid, a cyclic analogue of glutamic acid, the increase in c G M P was also lower in the older rats (Fig. 2). Similarly, the kainate-induced elevation of c A M P was markedly less in the cerebella of 12- or 24month-old rats (Fig. 3). Mao 19 proposed that c G M P synthesis occurs in the Purkinje cells in response to glutamate stimulation, and part of the norepinephrine-induced elevation of c A M P in
173 1OO0
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Fig. 3. Basal and kainate-stimulated accumulation of cyclic AMP in cerebellar slices from young, old and aged rats. Cerebellar slices from individual rats were prepared and incubated as indicated in Fig. 1. After 2 h 15 min of preincubation, kainate was added to the incubation medium and the incubation continued for 15 min. Values represent the mean ± S.E.M. of 10-13 individual determinations, each of which was assayed in duplicate for cAMP concentration. Values which differ significantl) from the 3-month-old rats are indicated (*); P < 0.05, Student's t-test.
the cerebellum occurs in Purkinje cells 35. The Purkinje cells also serve as the efferent system for the cerebellum. A n y loss of Purkinje cells with aging might have a dramatic effect on b o t h the biochemistry and function of the cerebellum. The cerebellum is one area where there have been confirmatory reports indicating a loss in cells. Purkinje cell loss with age has been seen in h u m a n s 7 and other vertebrates n. A loss of Purkinje cells during aging would explain the reduced elevation o f c G M P in cerebellar slices of aged rats (Fig. 2) and the reduction in c A M P synthesis in response to norepinephrine (Fig. 1) or in the presence o f the glutamate analogue kainic acid (Fig. 3). In support, we f o u n d that in 'nervous' m u t a n t mice, which have only 10 ~ o f the normal complement o f Purkinje cells 34, there was a reduction in b o t h basal and low level kainatestimulated cyclic nucleotide synthesis in vitro zv. However, it appears that other cerebellar elements besides the Purkinje cells can also synthesize cyclic nucleotides at
174 3 MONTH 12 120
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DOPAMINE
Fig. 4. Dopamine stimulation of cyclic AMP accumulation in homogenates of the corpus striatum from young, old and aged rats. The striatum from individual rats was dissected and homogenized as indicated in Methods. Adenylate cyclase activity was estimated by monitoring the conversion of [asP]aATP to [3zP]cAMP during 3 min incubations. Values represent the mean per cent increases in cAMP synthesis ± S.E.M. of 9 individual determinations, each of which was assayed in duplicate for adenylate cyclase activity. Values which differ significantly from the 3-month-old rats are indicated (*); P < 0.05, Student's t-test. Basal activities did not differ significantly with age: 3 month - - 162.8 ± 18.7; 12 month - - 148.7 zk 22.7; 24 month - - 120.3 i 15.2 pmoles of cAMP synthesized/mg protein/min at 37 °C. higher levels o f stimulation 27. This would explain why the greatest decrements (70 reductions) occurred at the lower concentration of kainate (Figs. 2 and 3). A reduction in the concentration and synthesis of dopamine in the corpus striatum of aged rats 8,z0 and humans 21 is t h o u g h t to account for some o f the m o v e m e n t disorders seen in the aged. Kebabian et al. 15 proposed that activation of striatal dopamine-sensitive adenylate cyclase is the means by which dopamine exerts its effects in the striatum. We studied dopamine stimulation of c A M P synthesis in homogenates of the corpus striatum, since dopamine does not stimulate c A M P accumulation in intact tissues (unpublished observations). Basal activity o f adenylate cyclase was approximately 23 ~ lower in striatum f r o m 24-month-old rats c o m p a r e d to 3-month-old controls: 120. 3~z 15.2 vs. 168.8 -4- 18.7 pmoles c A M P synthesized/mg protein/min, respectively. D o p a m i n e elevated c A M P synthesis at all ages, but the per cent stimulation was significantly lower in the older rats (Fig. 4). The slight differences in cylcase activity between the 12- and 24-month-old rats were not significant. Walker and Walker ~7 and Puri and Volcier 24 also measured catecholaminesensitive adenylate cyclase activity during aging, but they f o u n d that dopamine did n o t
175 activate cAMP synthesis in the striatum of 2-year-old rats. We observed that, although dopamine stimulation of cAMP synthesis in the striatum declined with age, dopamine was able to significantly elevate cAMP levels in tissue preparations from rats at all ages (Fig. 4). Our data derived from homogenates from 3-month-old rats agree with basal activity and the degree of dopamine stimulation of cAMP synthesis reported by others 3,15. Pharmacological studies with our system 2~ also agree with the findings of others in terms of the specificity of the dopamine response in this region. Therefore our methods seem technically correct. Furthermore, there are reasons to suspect that the results from the Walker and Walker experiments might be atypical : (1) the biphasic dopamine and norepinephrine dose-response relationships reported by Walker and Walker 37 have not been seen by us or other investigators measuring adenylate cyclase activity in the striatum; (2) anesthetized rats were used in the studies; (3) norepinephrine inhibition of striatal adenylate cyclase has not been observed by us or others; and (4) homogenates were frozen prior to assay, which has been shown to partially reduce dopamine stimulation of cyclic AMP synthesis 3. The inability of Puri and Volcier 24 to detect a significant dopamine-induced elevation of striatal adenylate cyclase activity in old rats might reflect the fact that in the young animals the degree of stimulation by dopamine was only 55 ~ above control. In our experiments (Fig. 4) and most others3,15, z5 dopamine at 10-4 M causes over 100 ~ increase in activity. Since the basal enzyme activity published by Puri and Volcier 24 is similar to that we have reported, it appears that a maximal response to dopamine-receptor stimulation is not occurring in their system. This is probably due to their use of a high concentration of Mg 2~ (5 mM) which tends to reduce dopamine activation of adenylate cyclase in the striatum 3. The 20 ~ decline in dopamine-stimulated cAMP synthesis in the striatum with aging indicates that postsynaptic receptor systems partially fail in this area. That caudate tyrosine hydroxylase and dopamine levels are lower in aged rats 2°,s indicates that presynaptic elements are also affected. Such changes in the dopaminergic transmitter system in this region probably contribute significantly to the functional declines which are observed in aged individuals, and may explain the reduced sensitivity of aged rats 36 and humans 22 to the stimulatory effects of amphetamine. The present experiments indicate that the cyclic nucleotide generating systems in the cerebellum and corpus striatum might be especially vulnerable to the effects of aging. No significant changes in resting or stimulated cyclic nucleotide accumulation were observed in the cortex, hippocampus, hypothalamus or brain stem. Nonetheless, intracellular alterations at points beyond cyclic nucleotide accumulation might occur and lead to tissue dysfunction. This appears to be the case in blood vessels where the age-related decline in the ability to relax in response to epinephrine 9 appears not to be due to a reduced capacity to synthesize cAMP 4. For this reason we are presently examining cAMP-dependent protein kinase activity in brain regions from aged rats and studying the phosphorylation patterns in these tissues. We thank Mr. Leo Hill and John Ryan for their technical assistance, and Dr. Ray Fuller for his critical reading of the manuscript.
176 1 Berg, A. and Zimmerman, I. D., Effects of electrical stimulation and norepinephrine on cyclicAMP levels in the cerebral cortex of the aging rat, Mech. Aging Dev., 4 (1975) 377-383. 2 Bitensky, M. W., Russell, V. and Blanco, M., Independent variation of glucagon and epinephrine responsive components of hepatic adenylcyclase as a function of age, sex and steroid hormones, Endocrinology, 86 (1970) 154-159. 3 Bockart, J., Premont, J., Glowinski, J., Thierry, A. M. and Tassin, J. P., Topographical distribution of dopaminergic innervation and of dopaminergic receptors in the rat striatum. II. Distribution and characteristics of dopamine adenylate cyclase-interaction with dopaminergic receptors, Brain Research, 107 (1976) 303-315. 4 Cohen, M. L. and Berkowitz, B. A., Age-related changes in vascular responsiveness to cyclicnucleotides and contractile agonists, J. Pharmacol. exp. Ther., 191 (1974) 147-155. 5 Daly, J., Role of cyclic nucleotides in the nervous system. In L. L. Iversen, S. D. Iversen and S. H. Snyder (Eds.), Handbook ofPsyehopharmacology, Vol. 5, Plenum Publishing, New York, 1975, p. 47-130. 6 Dayan, A. D., Comparative neuropathology of ageing--studies on the brains of 47 species of vertebrates, Brain, 94 (1971) 3142. 7 Ellis, R. S., Norms for some structural changes in the human cerebellum from birth to old age, J. eomp. Neurol., 32 (1930) 1-33. 8 Finch, C. E., Catecholamine metabolism in the brains of aging male mice, Brain Research, 52 (1973) 261-276. 9 Fleisch, J. H., Maling, H. M. and Brodie, B. B., Beta-receptor activity in aorta: variations with age and species, Circ. Res., 26 (1970) 151-162. 10 Forn, J., Schonhofer, P. S., Skidmore, I. F. and Kirshna, G., Effect of aging on the adenyl cyclase and phosphodiesterase activity of isolated fat cells of rat, Bioehem. biophys. Acta (Amst.), 28 (1970) 304-309. 11 Geinesman, Y. and Bondareff, W., Decrease in the number of synapses in the senescent brain: a quantitative electron microscopic analysis of the dentate gyrus molecular layer in the rat, Mech. Ageing Dev., 5 (1976) 11-23. 12 Gilman, A. G., A protein binding assay for adenosine 3',5'-cyclic monophosphate, Proc. nat. Aead. Sci. (Wash.), 67 (1970) 305-312. 13 Glowinski, J. and Iversen, L. L., Regional studies of catecholamines in the rat brain. I. The disposition of [3H]norepinephrine, [3H]dopamine and [3H]DOPA in various regions of the brain, J. Neurochem., 13 (1966) 655-669. 14 Gold, P. E. and McGaugh, J., Changes in learning and memory during aging. In J. M. Ordy and K. R. Brizzee (Eds.), Advances in BehavioralBiology, VoL 16, Plenum New York, 1975, pp. 145-158. 15 Kebabian, J. W., Petzold, G. L. and Greengard, P., Dopamine-sensitive adenylate cyclase in caudate nucleus of rat brain, and its similarity to the 'dopamine receptor', Proe. nat. Aead. Sci. (Wash.), 69 (1972) 2145-2149. 16 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 17 Makman, M. H., Properties of adenylate cyclase of lymphoid cells, Proe. nat. Aead. Sci. (Wash.), 68 (1971) 885-889. 18 Manganiello, V. and Vaughan, M., Selective loss of adipose cell responsiveness to glucagon with growth in the rat, J. Lipid Res., 13 (1972) 12-16. 19 Mao, C. C., Guidotti, A. and Costa, E., The regulation of cyclic guanosine monophosphate in rat cerebellum: possible involvement of putative amino acid neurotransmitters, Brain Research, 79 (1974) 510-514. 20 McGeer, E. G., Fibiger, H. C., McGeer, P. L. and Wickson, V., Aging and brain enzymes, Exp. GerontoL, 6 (1971) 391-396. 21 McGeer, E. and McGeer, P. L., Neurotransmitter metabolism in the aging brain. In R. D. Terry and S. Gershon (Eds.), Neurobiology of Aging, Raven Press, New York, 1976, pp. 389-403. 22 Nathanson, M. H., The central action of beta-aminopropylbenzene (benzedrine), J. Amer. med. Ass., 108 (1937) 528-531. 23 Palmer, G. C., Sulser, F. and Robison, G. A., Effects of neurohumoral and adrenergic agents on cyclic AMP levels in various areas of the rat brain in vitro, Neuropharmaeology, 12 (1973) 327-337. 24 Purl, S. K. and Volicer, L., Effect of aging on cyclic AMP levels and adenylate cyclase and phosphodiesterase activities in the rat corpus striatum, Mech. Ageing Dev., 6 (1977) 53-58. 25 Schmidt, M. J. and Hill, L. E., Effects of ergots on adenylate cyclase activity in the corpus str~atum
177 and pituitary, Life Sci., 20 (1977) 789-798. 26 Schmidt, M. J., Hopkins, J. T., Schmidt, D. E. and Robison, G. A., Cyclic AMP in brain areas: effects of amphetamine and norepinephrine assessed through the use of microwave radiation as a means of tissue fixation, Brain Research, 42 (1972) 465-477. 27 Schmidt, M. J. and Nadi, N. S., Cyclic nucleotide accumulation in vitro in the cerebellum of 'nervous' neurologically mutant mice, J. Neurochem., 29 (1977) 87-90. 28 Sehmidt, M. J., Palmer, E. C., Dettbarn, W.-D. and Robison, G. A., Cyclic AMP and adenyl cyclase in the developing rat brain, Develop. Psyehobiol., 3 (1970) 53-67. 29 Schmidt, M. J. and Robison, G. A., The effect of norepinephrine on cyclic AMP levels in discrete regions of the developing rabbit brain, Life Sci., 10 (1971) 459-464. 30 Schmidt, M. J. and Robison, G. A., The effect of neonatal thyroidectomy on the development of the adenosine 3',5'-monophosphate s~,stem in the rat brain, J. Neurochem., 19 (1972) 937-947. 31 Schmidt, M. J., Ryan, J. J. and Molloy, B. B., Effects of kainic acid, a cyclic analogue of glutamic acid, on cyclic nucleotide accumulation in slices of rat cerebellum, Brain Research, 112 (1976) 113-126. 32 Schmidt, M. J., Schmidt, D. E. and Robison, G. A., Cyclic adenosine monophosphate in brain areas: microwave irradiation as a means of tissue fixation, Science, 173 (1971) 1142-1143. 33 Sheppard, H. and Burghardt, C. R., Age-dependent changes in the adenylate cyclase and phosphodiesterase activity of rat erythrocytes, Biochem. Pharmacol., 22 (1973) 427-429. 34 Sidman, R. L. and Green, M. C., 'Nervous', a new mutant mouse with cerebellar disease. In M. Sadourdy (Ed.), Les Mutants Pathologiques Chez l'Animal, C.N.S.R., Paris, 1970, pp. 69-79. 35 Siggins, G. R., Battenberg, E. F., Hoffer, B. J., Bloom, F. E. and Steiner, A. L., Noradrenergic stimulation of cyclic adenosine monophsophate in rat purkinje neurons : an immunocytochemical study, Science, 179 (1973) 585-588. 36 VerzS_r, F., The age of the individual as one of the parameters of pharmacological action, Acta physiol, hung., 19 (1961) 313-318. 37 Walker, J. B. and Walker, J. P., Properties of adenylate cyclase from senescent rat brain, Brain Research, 54 (1973) 391-396. 38 Weiss, B. and Strada, S. J., Adenosine 3',5'-monophosphate during fetal and postnatal development. In L. Bor6us (Ed.), FetalPharmacology, Raven Press, New York, 1973, pp. 205-235. 39 Zimmerman, I. and Berg, A., Levels of adenosine 3',5'-cyclic monophosphate in the cerebral cortex of aging rats, Mech. Ageing Dev., 3 (1974) pp. 33-36.
169
Cyclic AMP and cyclic GMP accumulation in vitro in brain regions of young, old and ,aged rats
MICHAEL J. SCHMIDT* and JOHN F. THORNBERRY Lilly Research Laboratories, Eli Lilly and Company Indianapolis, Ind. 46206 (U.S.A.)
(Accepted August 8th, 1977)
Ultrastructural changes in synaptic morphology 11 or abnormalities in neurotransmitter-receptor interaction might account for the functional defects often seen in the aged. Neurotransmitter-stimulated synthesis of cyclic nucleotides in brain slices has been widely used as a biochemical measure of receptor activity 5, and the ability of norepinephrine to stimulate adenosine 3'5'-monophosphate (cAMP) synthesis in brain tissue is age-dependent. Norepinephrine does not elevate cAMP levels in brain slices until 5-6 days after birth zs, even though adenylate cyclase is present in the rat brain at birth. A similar phenomenon was seen in the rabbit brain, but in this species a marked reduction in hormone-stimulated c A M P synthesis occurred in old animals 29. The latter studies suggested that a reduction in transmitter-stimulated c A M P accumulation might contribute to the mental dysfunction which often accompanies old age. The purpose of the present studies was to survey transmitter-receptor interactions in young, old and aged rats by monitoring norepinephrine-stimulated accumulation of cyclic nucleotides in slices of brain regions incubated in vitro. Rats older than 3 months of age were obtained from a colony of aged Wistar rats maintained by Harlan Industries, Indianapolis, Ind. Three-month-old rats came from general Harlan stock. All rats were maintained under conditions which meet or surpass AALAS standards. Rats in the colony were examined daily and any animal with demonstrable abnormalities (tumors, blindness, excessive weight loss) was eliminated. A 12-h light-dark cycle was in effect at all times. Animals were fed and watered ad libitum. The ages chosen for study were based on the suggestion of Finch s that mice at the age of average longevity are comparable to human males 70-80 years old. Twentyfour months of age represents the 50 ~ survival level in our colony and was selected as the upper end of the age scale. Three-month-old rats were selected as the youngest group since at this age rats are sexually mature and past the stage of rapid brain growth. A 12-month-old group was included to give some indication whether our
* To whom correspondence should be addressed.
170 findings were a reflection of aging (12 months vs. 24 months), maturation (3 months vs. 12 months) or an aging continuum (3 vs. 12 vs. 24 months). Male Wistar rats were killed by decapitation, the brains removed and dissected la and brain areas minced on an ice-cooled plate. Cyclic nucleotide accumulation was determined in chopped tissue slices as described previously 3a. It was necessary to preincubate some regions longer than others in order to achieve stable basal levels of cAMP: i.e. brain stem, hypothalamus and hippocampus - - 30 rain; cerebral cortex --- 60 min; c e r e b e l l u m - 120 rain. Purification of cyclic nucleotides by sequential alumina and Dowex 1 chromatography, and separation of cAMP and guanosine 3',5'monophosphate (cGMP) by clution with 0.05 N HC1 and 0.5 N HC1, respectively, has been described 31. Recovery of cAMP was approximately 60 ~ and c G M P approximately 40 ~ using this method. Assay values of cyclic nucleotide concentrations were corrected for this loss during purification. Final results are expressed as pmoles of cyclic nucleotide per mg protein per incubation time. Adenylate cyclase activity was determined by measuring the conversion of [32p]aATP to [3ZP]cAMP and separating and purifying the end-product by the dual column procedure outlined above 25. Cyclic AMP concentrations from tissue slice experiments were determined using a protein binding assay 12, and c G M P levels by immunoassay (Schwartz-Mann, Orangeburg, N.Y.). Protein concentration was measured by the method of Lowry et al. 16 using bovine serum albumin as the standard. Kainic acid was obtained from Sigma Chemical (St. Louis, Mo.). The remaining chemicals and supplies were purchased from other commercial sources. Norepinephrine stimulated cAMP accumulation in all brain areas at all ages although the degree of stimulation varied across regions (Fig. 1). The cerebellum was the only area in which a significant age-related difference in cAMP accumulation was observed in tissue slices. The norepinephrine-elicited elevation in cAMP was 44 lower in the cerebellum of 12-month-old rats and 57 ~ lower in 24-month-old rats in comparison to 3-month-old animals. Resting levels of cAMP were also lower in slices of the cerebellum from 24-month-old rats, but in all other regions basal levels of cAMP were similar across ages. Age-related declines in basal and stimulated cAMP accumulation have been observed in adipose tissue10, is, lymphocytes iv, erythrocytes 3a and the liver 2. We demonstrated that norepinephrine-stimulated cAMP accumulation was higher in whole rat brain at 15 days of age than at 25 days of age 3°, and Weiss and Strada as found similar decreases in adenylate cyclase activity in several brain regions between 15 and 60 days of age. All of these studies reveal age-related declines in hormonereceptor interaction, but the differences actually reflect maturation rather than aging for in no instance were animals used which approached the two year point which approximates the senescent condition in rats. The present findings indicate that there is not a progressive decline in the concentration of cAMP in unstimulated tissue slices from the hypothalamus, cerebral cortex, hippocampus, or brain stem as young mature Wistar rats grow into old age. Puri and Volicer 24 also found no change in cAMP levels in the striatum of aged rats. At variance with our results are the findings of Zimmerman and Berg1, a°. They
171 observed a significant reduction in cortical c A M P in Fisher 344 rats at 6 m o n t h s o f age when c o m p a r e d to 3 - m o n t h - o l d rats. H o w e v e r , again this difference seems to represent a m a t u r a t i o n a l change for there was no further decline in c A M P levels during the last 18 m o n t h s o f the animal's life when the behavioral manifestations o f aging are seen 14. Berg and Z i m m e r m a n 1 also r e p o r t e d that in older rats there was less accumulation o f c A M P in cortical tissues which had been exposed to n o r e p i n e p h r i n e in situ. O u r results are in disagreement with these findings. It is possible the strain differences (Fisher 344 vs. Wistar) account for the disparity. H o w e v e r , m e t h o d o l o g i c a l differences
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Hypothalamus
Cerebellum
Fig. 1. Basal and norepinephrine-stimulated accumulation of cyclic AMP in slices of brain regions from young, old and aged rats. Brain regions from individual rats were minced, rinsed with KrebsRinger buffer and preincubated in 20 ml of buffer for varying times at 37 °C as indicated in Methods. Then the medium was replaced with 9.9 ml of fresh, cold medium, and 15 min later 0.1 ml of an aqueous solution of norepinephrine or water alone was added to eadh flask, such that the final concentration of norepinephrine was 10-5 M (except the cerebellum where 10-4 M norepinephrine was used). The reaction was terminated by adding perchloric acid and homogenizing the tissues at the time of maximal cAMP accumulation in each respective area: brain stem and hippocampus - - 6 rain; hypothalamus - - 10 rain; cerebellum - - 15 min; and cerebral cortex - - 20 min. Cyclic AMP was purified by sequential alumina and Dowex 1 chromatography. Values represent the mean pmoles cAMP/rag protein :~ S.E.M. Solid bars depict cyclic AMP concentrations in the absence of norepinephrine. The variation in basal levels of cAMP within groups was approximately 10 ~ .
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Fig. 2. Basal and kainate-stimulated accumulation of cyclic GMP in cerebellar slices from young, old and aged rats. Cerebellar slices from individual rats were prepared and incubated as indicated in Fig. 1. After 2 h 15 min of preincubation, kainate was added to the incubation medium and the incubation continued for 15 min. Values represent the mean ± S.E.M. of 11-13 individual determinations, each of which was assayed in duplicate for cGMP concentration. Values which differ significantly from the 3-month-old rats are indicated (*); P < 0.05, Student's t-test. between our studies and those of Zimmerman and Berg might also explain the lack of agreement. For example, Berg and Zimmerman 1 estimated hormone stimulation by adding norepinephrine to cotton pledgets placed on the cortex of anesthetized rats. Dose-response relationships cannot be controlled under these conditions and previous studies have shown that anesthesia reduces cAMP levels in vivo 26. Furthermore, the rats were sacrificed by decapitation prior to tissue fixation and this produces differences in c A M P concentrations in all brain regions z~. In our experiments the basal and norepinephrine-stimulated levels of c A M P observed in cortical slices from 3month-old rats (Fig. 1) are comparable to the work of others zz. Cyclic G M P levels were determined in brain regions during control incubations. The level o f c G M P was much lower than c A M P in most all areas: cerebral cortex (0.31 ~- 0.05), hippocampus (0.45 ~: 0.06), hypothalamus (0.44 ± 0.03) and brain stem (0.42 z~ 0.08 pmoles/mg protein). Concentrations were similar at 3, 12 and 24 months of age in these areas. However, in the cerebellum the concentration of c G M P was significantly lower at 24 months of age than at 12 or 3 months (Fig. 2). When cerebellar slices were stimulated by the addition of kainic acid, a cyclic analogue of glutamic acid, the increase in c G M P was also lower in the older rats (Fig. 2). Similarly, the kainate-induced elevation of c A M P was markedly less in the cerebella of 12- or 24month-old rats (Fig. 3). Mao 19 proposed that c G M P synthesis occurs in the Purkinje cells in response to glutamate stimulation, and part of the norepinephrine-induced elevation of c A M P in
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Fig. 3. Basal and kainate-stimulated accumulation of cyclic AMP in cerebellar slices from young, old and aged rats. Cerebellar slices from individual rats were prepared and incubated as indicated in Fig. 1. After 2 h 15 min of preincubation, kainate was added to the incubation medium and the incubation continued for 15 min. Values represent the mean ± S.E.M. of 10-13 individual determinations, each of which was assayed in duplicate for cAMP concentration. Values which differ significantl) from the 3-month-old rats are indicated (*); P < 0.05, Student's t-test.
the cerebellum occurs in Purkinje cells 35. The Purkinje cells also serve as the efferent system for the cerebellum. A n y loss of Purkinje cells with aging might have a dramatic effect on b o t h the biochemistry and function of the cerebellum. The cerebellum is one area where there have been confirmatory reports indicating a loss in cells. Purkinje cell loss with age has been seen in h u m a n s 7 and other vertebrates n. A loss of Purkinje cells during aging would explain the reduced elevation o f c G M P in cerebellar slices of aged rats (Fig. 2) and the reduction in c A M P synthesis in response to norepinephrine (Fig. 1) or in the presence o f the glutamate analogue kainic acid (Fig. 3). In support, we f o u n d that in 'nervous' m u t a n t mice, which have only 10 ~ o f the normal complement o f Purkinje cells 34, there was a reduction in b o t h basal and low level kainatestimulated cyclic nucleotide synthesis in vitro zv. However, it appears that other cerebellar elements besides the Purkinje cells can also synthesize cyclic nucleotides at
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Fig. 4. Dopamine stimulation of cyclic AMP accumulation in homogenates of the corpus striatum from young, old and aged rats. The striatum from individual rats was dissected and homogenized as indicated in Methods. Adenylate cyclase activity was estimated by monitoring the conversion of [asP]aATP to [3zP]cAMP during 3 min incubations. Values represent the mean per cent increases in cAMP synthesis ± S.E.M. of 9 individual determinations, each of which was assayed in duplicate for adenylate cyclase activity. Values which differ significantly from the 3-month-old rats are indicated (*); P < 0.05, Student's t-test. Basal activities did not differ significantly with age: 3 month - - 162.8 ± 18.7; 12 month - - 148.7 zk 22.7; 24 month - - 120.3 i 15.2 pmoles of cAMP synthesized/mg protein/min at 37 °C. higher levels o f stimulation 27. This would explain why the greatest decrements (70 reductions) occurred at the lower concentration of kainate (Figs. 2 and 3). A reduction in the concentration and synthesis of dopamine in the corpus striatum of aged rats 8,z0 and humans 21 is t h o u g h t to account for some o f the m o v e m e n t disorders seen in the aged. Kebabian et al. 15 proposed that activation of striatal dopamine-sensitive adenylate cyclase is the means by which dopamine exerts its effects in the striatum. We studied dopamine stimulation of c A M P synthesis in homogenates of the corpus striatum, since dopamine does not stimulate c A M P accumulation in intact tissues (unpublished observations). Basal activity o f adenylate cyclase was approximately 23 ~ lower in striatum f r o m 24-month-old rats c o m p a r e d to 3-month-old controls: 120. 3~z 15.2 vs. 168.8 -4- 18.7 pmoles c A M P synthesized/mg protein/min, respectively. D o p a m i n e elevated c A M P synthesis at all ages, but the per cent stimulation was significantly lower in the older rats (Fig. 4). The slight differences in cylcase activity between the 12- and 24-month-old rats were not significant. Walker and Walker ~7 and Puri and Volcier 24 also measured catecholaminesensitive adenylate cyclase activity during aging, but they f o u n d that dopamine did n o t
175 activate cAMP synthesis in the striatum of 2-year-old rats. We observed that, although dopamine stimulation of cAMP synthesis in the striatum declined with age, dopamine was able to significantly elevate cAMP levels in tissue preparations from rats at all ages (Fig. 4). Our data derived from homogenates from 3-month-old rats agree with basal activity and the degree of dopamine stimulation of cAMP synthesis reported by others 3,15. Pharmacological studies with our system 2~ also agree with the findings of others in terms of the specificity of the dopamine response in this region. Therefore our methods seem technically correct. Furthermore, there are reasons to suspect that the results from the Walker and Walker experiments might be atypical : (1) the biphasic dopamine and norepinephrine dose-response relationships reported by Walker and Walker 37 have not been seen by us or other investigators measuring adenylate cyclase activity in the striatum; (2) anesthetized rats were used in the studies; (3) norepinephrine inhibition of striatal adenylate cyclase has not been observed by us or others; and (4) homogenates were frozen prior to assay, which has been shown to partially reduce dopamine stimulation of cyclic AMP synthesis 3. The inability of Puri and Volcier 24 to detect a significant dopamine-induced elevation of striatal adenylate cyclase activity in old rats might reflect the fact that in the young animals the degree of stimulation by dopamine was only 55 ~ above control. In our experiments (Fig. 4) and most others3,15, z5 dopamine at 10-4 M causes over 100 ~ increase in activity. Since the basal enzyme activity published by Puri and Volcier 24 is similar to that we have reported, it appears that a maximal response to dopamine-receptor stimulation is not occurring in their system. This is probably due to their use of a high concentration of Mg 2~ (5 mM) which tends to reduce dopamine activation of adenylate cyclase in the striatum 3. The 20 ~ decline in dopamine-stimulated cAMP synthesis in the striatum with aging indicates that postsynaptic receptor systems partially fail in this area. That caudate tyrosine hydroxylase and dopamine levels are lower in aged rats 2°,s indicates that presynaptic elements are also affected. Such changes in the dopaminergic transmitter system in this region probably contribute significantly to the functional declines which are observed in aged individuals, and may explain the reduced sensitivity of aged rats 36 and humans 22 to the stimulatory effects of amphetamine. The present experiments indicate that the cyclic nucleotide generating systems in the cerebellum and corpus striatum might be especially vulnerable to the effects of aging. No significant changes in resting or stimulated cyclic nucleotide accumulation were observed in the cortex, hippocampus, hypothalamus or brain stem. Nonetheless, intracellular alterations at points beyond cyclic nucleotide accumulation might occur and lead to tissue dysfunction. This appears to be the case in blood vessels where the age-related decline in the ability to relax in response to epinephrine 9 appears not to be due to a reduced capacity to synthesize cAMP 4. For this reason we are presently examining cAMP-dependent protein kinase activity in brain regions from aged rats and studying the phosphorylation patterns in these tissues. We thank Mr. Leo Hill and John Ryan for their technical assistance, and Dr. Ray Fuller for his critical reading of the manuscript.
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