Histochemistry51, 113- 119 (1977)
Histochemistry 9 by Springer-Verlag 1977
Electron Microscopical Demonstration of Adenylate Cyclase Activity in Nervous Tissue Leena Rechardt and Matti Hfirk6nen Department of Anatomy,Universityof Helsinki, SF-00170 Helsinki 17 and Department of ClinicalChemistry,Universityof Helsinki, MeilahtiHospital, SF-00290 Helsinki29, Finland
Summary. Adenylate cyclase (EC 4.6.1.1) activity stimulated by norepinephrine and dopamine was demonstrated histochemically by electron microscopy in the cerebral cortex and caudate nucleus of the rat. The precipitating agent in the histochemical reaction was cobalt, which was shown biochemically to increase the adenylate cyclase activity. The reaction product was located in the synapses, being contiguous attached to the postsynaptic membrane. It was also located in the plasma membrane of some nerve fibers. Alloxan, the specific inhibitor of adenylate cyclase, inhibited the reaction in the cerebral cortex and caudate nucleus, and haloperidol had a somewhat similar effect in the caudate region. Introduction
Cyclic adenosine 3',5'-monophosphate (cAMP) is known to be involved in neural transmission, and brain tissue is one of the richest sources of the enzyme adenylate cyclase (von Hungen and Roberts, 1974; Perkins, 1973). Adenylate cyclase is in the brain activated by neurotransmitters, e.g. by norepinephrine in the cerebral cortex (Huang et al., 1971) and by dopamine in the caudate nucleus (Kebabian et al., 1972). Biochemically, the activity of this enzyme seems to be related to the microsomal fraction, and especially to the synaptosomes, as shown by De Robertis et al. (1967). Hitherto, there have been no reports of histochemical demonstration of adenylate cyclase activity in nervous tissue. Using lead as a trapping agent (Wagner and Bitensky, 1974), several investigators have claimed to demonstrate adenylate cyclase activity in other mammalian tissues, such as liver cells (Reik et al., 1970), the islets of Langerhans (Howell and Whitfield, 1972) the capillary endothelium (Wagner et al., 1972) the nephron (Jande and Robert, 1974), and the uterine epithelium (Sananes and Psychoyos, 1974). Recently, however, serious criticism has been leveled at the use of the lead technique for the demonstration of adenylate cyclase * Supported by grants from the MedicalResearch Councilin the Academyof Finland
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activity (Lemay and Jarett, 1975). Lemay and Jarett (1975) using a biochemical method, have shown that lead, at the concentrations employed in the above studies, totally inhibits adenylate cyclase activity. The present paper describes a method in which cobalt is used as a precipitating agent for demonstrating adenylate cyclase activity in nervous tissue. Cobalt salts are visible in the electron microscope, and, moreover, Co + + was found to stimulate adenylate cyclase activity.
Material and Methods Adult male albino rats of Sprague-Dawley strain weighing about 250 g were used for the study. The experimental rats were injected with L-dopa (L-3,4-dihydroxyphenylalanine, L-dopa. Orion Oy, Finland) 100 nag kg/body wt. intraperitoneally daily during one week, and then killed with a sharp blow on the neck. The controls were given physiological saline. The anterior parts of the cortex and eaudate nuclei of the brain were quickly removed on chilled ice, and cut into thin slices with a razor blade or a tissue chopper (The Mickle Laboratory Engineering Co, England). The slices of brain cortex to be used for histochemical demonstration were pre-incubated at 37~ C for 20 rain in a medium (modified Krebs-Ringer-glucose solution) (Larrabee, 1958) buffered with Tris (hydroxymethyl) methyl-glycine (TRICINE, Sigma, St. Louis, Mo.) containing 1-3 retool/1 adenine (E. Merck AG, Darmstadt), or 1 3 mmol/1 adenosine (E. Merck AG, Darmstadt) together with 0.1 retool/1 norepinephrine hydrochloride (Fluka AG, Buchs SG). For the slices of caudate nuclei, the same medium was used, with the addition of 3 mmol/1 dopamine (Dopamin-hydrochlorid, Fluka AG, Buchs SG). The slices were then fixed in freshly prepared 1% paraformaldehyde-KrebsRinger-glucose (Larrabee, 1958) buffered with TRICINE to pH 7.0. After fixation for 5 min at 4~ C the slices were washed for 30 min in three changes of Krebs-Ringer-glucose solution. Some of the cortex slices were processed further without any prefixation. Histochemical incubation was carried out for 30 min at room temperature (22~ C) in a medium with the final concentration: TRICINE buffer (pH 8.3) 50 retool/l, MgCI2 3 mmol/1, CaC1/8 mmol/1, Co (NO3)2 3 retool/l, ATP disodium salt (Fluka AG, Bucks SG) 3 mmol/1, ouabain 1 mmol/I, theophylline 10 retool/l, and L-tetramisole (R 12564, Janssen Pharmaceutica, Netherlands) 0.5 mmol/ 1. The pH of the solution was adjusted to 8.3. The solution was clear and light pinkish in color. After incubation the slices were washed for 30 min in three changes of ice-cold physiological saline, post-fixed in 1% OsO4 for 1 h, dehydrated, and embedded in Epon-Aratdite mixture. The ultrathin sections were cut with diamond knives and viewed and photographed unstained with a Philips EM 300, at 40 kV. In preliminary studies the concentrations of all the reagents were varied over a wide range. In the control studies cobalt, ATP and inhibitors were omitted. ATP was replaced with/3-glycerophosphate (E. Merck AG, Darmstadt). The slices were heated at 100~ C for 10 min. In the preliminary studies the incubation media also included 2 retool/1 dithiotreitol or 10 retool/1 NaF, but because these compounds formed visible precipitates in the incubation solutions their use was abondoned. In some incubation solutions 5 retool/1 alloxan hydrate (Fluka AG, Buchs) was used. Some rats were given injections of haloperidol (Orion, Helsinki) 10 mg/kg/body wt. intraperitoneally 24 h before they were killed. The biochemical control assays of adenylate cyclase activity were performed with homogenates of cortical slices. The enzyme activity was measured after stimulation with adenosine-norepinephrine and the value compared with that for unstimulated control slices. Adenosine triphosphatase activity was checked from the cortical slices under the incubation conditions using the method by H/irk6nen et al. (1972). No biochemical activity was detected. Comparative values of adenylate cyclase activity were also measured after fixation for 5 min in 1% paraformaldehyde solution; likewise, the effect of 3 mmol/1 Co (NO3)2 in the Krebs-Ringer-glucose was tested. For details of the method used for measuring the activity of the enzyme see Hfirk6nen et al. (1974).
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Fig. 1. Dopamine-stimulated adenylate cyclase activity in the caudate nucleus of the rat. The reaction product is found in the plasma membrane of the large nerve fiber. Two terminal axons with synaptic vesicles are seen and the postsynaptic membrane exhibits enzyme activity (arrows). The neighboring nerve fibers show no visible enzyme activity. The b a r = 0 . 5 gm in all figures, x 13,200
Fig. 2. Norepinephrine-stimulated adenylate cyclase activity in the cerebral cortex of rat brain. The reaction product is seen at the postsynaptic site in the postsynaptic membrane (arrow). The myelinated nerve fiber and mitochondria (34) are negative, x 19,800
116
L. Rechardt and M. H/irk6nen Fig. 3. Dopamine-stimulated adenylate cyciase activity in the caudate nucleus of the rat. The reaction product seen in the postsynaptic membrane is identical to that seen in the cerebral cortex. The pre-synaptic part and the synaptic vesicles are devoid of any reaction products, x 13,200 Fig. 4. A control section of the cerebral cortex without any preincubation with amines. No reaction product due to adenosine triphosphatase activity is observed in mitochondria or in myelinated nerve fibers, which are the common localization sites of adenosine triphosphatase. • 13,200 Fig. 5. A control section of the cerebral cortex. Five mmol/1 alloxan hydrate was included in the incubation medium. Alloxan hydrate totally inhibited the enzymatic reaction in the slices, x 13,200
Observations F i x a t i o n for 5 m i n in 1% p a r a f o r m a l d e h y d e s o l u t i o n gave g o o d p r e s e r v a t i o n o f the overall u l t r a s t r u c t u r e o f b r a i n tissue, a n d especially o f the s y n a p t i c structures, including the s y n a p t i c vesicles. M i t o c h o n d r i a were s o m e t i m e s swollen a n d extracellular spaces widened. In sections e x a m i n e d w i t h o u t a n y prefixation, the nerve fibers, synapses a n d m i t o c h o n d r i a were s o m e w h a t inferior to I those in fixed slices b u t still identifiable. Brief fixation with 1% p a r a f o r m a l d e h y d e was preferred, however, to m a k e h a n d l i n g o f the tissue slices easier. Biochemically, this fixation p r o c e d u r e d i m i n i s h e d the s t i m u l a t e d a d e n y l a t e cyclase activity only 9 % as c o m p a r e d with the activity o f unfixed cortical slices otherwise t r e a t e d identically. W i t h o u t a d e n o s i n e - n o r e p i n e p h r i n e p r e i n c u b a t i o n , which i n c r e a s e d the enz y m e activity b y 23%, the scanty, r a n d o m l y d i s t r i b u t e d p r e c i p i t a t e ( p r o b a b l y
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due to the basal activity of adenylate cyclase) was not found in any specific structures (Fig. 4). It is certain that the precipitate formed did not represent adenosine triphosphatase activity, because the mitochondria and myelinated nerve fibres were devoid of the reaction product (Fig. 4). This was also confirmed biochemically. The reaction product which was easily visible in the electron microscope, was localized around some nerve fibers in both the cerebral cortex and the caudate nucleus (see Fig. 1). Two nerve terminals seem to synapse on this nerve fiber and the reaction product is seen continuously at the synaptic sites. In some of the synapses the reaction product was attached contiguous to the postsynaptic membrane (Fig. 2 and 3). The presynaptic site did not show any activity. The morphology, distribution and number of synapses were not recorded in detail in this connection, but will be restudied in the future. When 5 mmol/1 alloxan hydrate was added to the incubation medium no precipitate was seen (Fig. 5). Similarly, in the caudate nucleus pretreatment with haloperidol reduced the number of synapses showing the reaction product. However, the enzyme activity was not as completely inhibited as would be expected on the basis of biochemical studies (Kebabian et al., 1972). When ATP was replaced with/%glycerophosphate no reaction product was seen with either tissue. The other adequate histochemical controls were also negative.
Discussion
Severe criticism has been leveled at the use of lead as a trapping agent for demonstrating phosphatases (Rosenthal et al., 1966) and adenylate cyclase (Lemay and Jarett, 1975). Glutaraldehyde fixation, used earlier for the histochemical demonstration of adenylate cyclase activity, has likewise been shown to destroy most of this activity (Wagner and Bitensky, 1974). In the present study, therefore, we used neither lead nor glutaraldehyde and made biochemical tests for any possible false-positive reactions due to the presence of phosphatases of various kinds. We therefore decided to use cobalt as a trapping agent. In biochemical tests we found that cobalt enhances adenylates cyclase activity by 16%. In alkaline solutions cobalt rapidly forms a hydroxylic precipitate with pyrophosphate. The cobalt salt is well visualized in the electron microscope, especially at low voltages, such as 40 kV. The most serious problem in demonstrating adenylate cyclase activity with this agent is the false-positive reactions due to phosphatases. The enzymes produce inorganic phosphate, which is also precipitated by cobalt. Sodium fluoride cannot be used to inhibit phosphatase, because it, too, forms a precipitate. We therefore tested several other ways to overcome this difficulty. Adenylate cyclase is active over a relatively broad pH range (von Hungen and Roberts, 1974; Clement-Cormier et al., 1975), and in the present study the upper limit of the pH range was adopted. At this pH the activities of adenosine phosphatases and nucleotidases are depressed, and in the incubation conditions used no adenosine triphosphatase activity was detectable biochemically in the brain slices.
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Even the mitochondria were as a rule negative. As a substrate, 5'-adenylylimidodiphosphate has been claimed to be a more specific for adenylate cyclase than ATP (Wagner and Bitensky, 1974). So far we have not used it, because in studies on localization with the lead techniques generally the two substrates gave identical results (see Wagner and Bitensky, 1974). Theophylline at a concentration as high as was used here inhibits phosphodiesterase activity (Cheung, 1970), which theoretically could interfere with the present method. Theophylline also prevents the further formation of monophosphates in the enzymatic cycle. Alloxan hydrate does not inhibit phosphodiesterase activity (Cohen and Bitensky, 1969), and as the alloxan controls were n~gative, it seems unlikely that phosphodiesterase was responsible for the activity demonstrated in the synapses by the present method. The other serious source of error might be the heterogeneous group of alkaline phosphatases, which could cause precipitates with ATP as substrate at pH 8.3. The introduction of a specific inhibitor of alkaline phosphatases by Borgers (1973) certainly has simplified the demonstration of phosphatases. In the present study no activity was observed with /?-glycerophosphate as substrate. Alloxan, moreover, does not inhibit either adenosine or alkaline phosphatases (Cohen and Bitensky, 1969) ; thus it presumably inhibited only the adenylate cyclase activities localized by the present technique. The paraformaldehyde fixation made it impossible to demonstrate the small dense-cored vesicles connected with aminergic transmission. The synapses contained only clear vesicles. It has been suggested that the ultrastructural localization of adenylate cyclase activity would demonstrate synapses containing norepinephrine receptors in the cerebral cortex and dopamine receptors in the caudate nucleus (Greengard et al., 1972). The postsynaptic histochemical localization observed in the present study confirms the results of biochemical measurements after pharmacological and denervation experiments, which showed that the adenylate cyclase connected with nervous transmission is located postsynaptically (Greengard et al., 1972). So far, no distinct presynaptic location has been observed in the brain tissue of the rat, although recently it has been proposed that adenylate cyclase is involved presynaptically in the synthesis of tyrosine hydroxylase and in microtubular function (Greengard, 1975). However, in cultured sympathetic ganglia of the chicken we have observed a presynaptic localization in the nerve fibers (Hervonen and Rechardt, in press), a finding which would confirm the hypothesis proposed by Greengard (1975). References Borgers, M.: The cytochemical application of new potent inhibitors of alkaline phosphatases. J. Histochem. Cytochem. 21, 812-824 (1973) Cheung, W.Y.: Cyclic nucleotide phosphodiesterase. In: Role of cyclic A M P in cell function (P. Greengard., E. Costa, ed.), Vol. 3, pp. 51 65. New York: Raven Press 1970 Clement-Cormier, Y.C., Parrish, R.G., Petzold, G.L., Kebabian, J.W., Greengard, P. : Characterization of a dopamine-sensitive adenylate cyclase in the rat caudate nucleus. J. Neurochem. 25, 143 149 (1975) Cohen, K.L., Bitensky, M.W. : Inhibitory effects of alloxan on mammalian adenyl cyclase. J. Pharmacol. exp. Ther. 169, 80-86 (1969)
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Greengard, P.: Prexynaptic and postsynaptic roles of cyclic AMP and protein phosphorylation at catecholaminergic synapses. In: Chemical tools in catecholamine research (O. Almgren., A. Carlsson., J. Engel, ed.), Vol. 2, pp. 249 256. Amsterdam: North-Holland Publishing Company 1975 Greengard, P., McAfee, D.A., Kebabian, J.W.: On the mechanism of action of cyclic AMP and its role in synaptic transmission. In: Advances in cyclic nucleotide research, Vol. 1, pp. 337 355. New York: Raven Press 1972 H~irkSnen, M., Hopsu-Havu, V.K., Raij, K.: Cyclic adenosine monophosphate, adenyl cyclase and cyclic nucleotide phosphodiesterase in psoriatic epidermis. Acta derm.-venerol. (Stockh.) 54, 13-18 (1974) Hfirk6nen, M., Palkama, A., Uusitalo, R. : Functional dependence of the ciliary epithelium ATPase activity and intraocular pressure on the autonomic nervous system. Acta physiol, scand. 86, 327 341 (1972) Hervonen, H., Rechardt, L. : Histochemical localization of adenylate cyclase in cultured sympathetic neurons. Histochemistry 48, 43-50 (1976) Howell, S.L., Whitfield, M. : Cytochemical localization of adenyl cyclase activity in rat islets of Langerhans. J. Histochem. Cytochem. 20, 873-879 (1972) Huang, M., Shimizu, J., Daly, J.: Regulation of adenosine cyclic 3',5'-phosphate formation in cerebral cortical slices, Molec. Pharmacol. 7, 155-162 (1971) Hungen, K. von, Roberts, S.: Neurotransmitter-sensitive adenylate cyclase systems in the brain. In: Reviews of neuroscience (S. Ehrenpreis., I. Kopin, ed.), Vol. l, pp. 231-281. New York: Raven Press 1974 Jande, S.S., Robert, P. : Cytochemical localization of parathyroid hormone activated adenyl cyclase in rat kidney. Histochemistry 40, 323-327 (1974) Kebabian, J.W., Petzold, G.L., Greengard, P.: Dopamine-sensitive adenylate cyclase in caudate nucleus of rat brain, and its similarity for the "dopamine receptor". Proc. nat. Acad. Sci. (Wash.) 69, 2145-2149 (1972) Larrabee, M.G.: Oxygen consumption of excised sympathetic ganglia at rest and in activity. J. Neurochem. 2, 81 10t (1958) Lemay, A., Jarett, L.: Pitfalls in the use of lead nitrate for the histochemical demonstration of adenylate cyclase activity. J. Ceil Biol. 65, 39-50 (1975) Perkins, J.P.: Adenyl cyclase. In: Advances in cyclic nucleotide research, Vol. 3, pp. 1~54. New York: Raven Press 1973 Reik, L., Petzold, G.L., Higgins, J.A., Greengard, P., Barrnett, R.J.: Hormone sensitive adenyl cyclase: cytochemical localization in the rat liver. Science 168, 382-384 (1970) DeRobertis, E., Arnaiz, G.R.D.L., Alberici, M., Butcher, R.W., Sutherland, E.W.: Subcellular distribution of adenyl cyclase and cyclic phosphodiesterase in rat brain cortex. J. biol. Chem. 242, 3487 3493 (1967) Rosenthal, A.S., Moses, H.L., Beaver, D.L., Schuffman, S.S. : Lead ion and phosphatase histochemistry. I. Non-enzymatic hydrolysis of nucleoside phosphates by lead ion. J. Histochem. Cytochem. 14, 698-701 (1966) Sananes, N., Psychoyos, A. : Cytochemical localization of adenyl cyclase in the rat uterus. J. Repr Fertil. 38, 181-183 (1974) Wagner, R.C., Bitensky, M.W. : Adenylate cyclase in electron microscopy of enzymes. In: Electt, microscopy of enzymes (M.A. Hayat, ed.), pp. 110-131. New York: Van Norstrand Company Inc. 1974 Wagner, R.C., Kreiner, P., Barrnett, R.J., Bitensky, M.W. : Biochemical characterization and cytochemical localization of a catecholamine-sensitive adenylate cyclase in isolated capillary endothelium. Proc. nat. Acad. Sci. (Wash.) 69, 3176-3179 (1972)
Received October 10, 1976
Histochemistry 9 by Springer-Verlag 1977
Electron Microscopical Demonstration of Adenylate Cyclase Activity in Nervous Tissue Leena Rechardt and Matti Hfirk6nen Department of Anatomy,Universityof Helsinki, SF-00170 Helsinki 17 and Department of ClinicalChemistry,Universityof Helsinki, MeilahtiHospital, SF-00290 Helsinki29, Finland
Summary. Adenylate cyclase (EC 4.6.1.1) activity stimulated by norepinephrine and dopamine was demonstrated histochemically by electron microscopy in the cerebral cortex and caudate nucleus of the rat. The precipitating agent in the histochemical reaction was cobalt, which was shown biochemically to increase the adenylate cyclase activity. The reaction product was located in the synapses, being contiguous attached to the postsynaptic membrane. It was also located in the plasma membrane of some nerve fibers. Alloxan, the specific inhibitor of adenylate cyclase, inhibited the reaction in the cerebral cortex and caudate nucleus, and haloperidol had a somewhat similar effect in the caudate region. Introduction
Cyclic adenosine 3',5'-monophosphate (cAMP) is known to be involved in neural transmission, and brain tissue is one of the richest sources of the enzyme adenylate cyclase (von Hungen and Roberts, 1974; Perkins, 1973). Adenylate cyclase is in the brain activated by neurotransmitters, e.g. by norepinephrine in the cerebral cortex (Huang et al., 1971) and by dopamine in the caudate nucleus (Kebabian et al., 1972). Biochemically, the activity of this enzyme seems to be related to the microsomal fraction, and especially to the synaptosomes, as shown by De Robertis et al. (1967). Hitherto, there have been no reports of histochemical demonstration of adenylate cyclase activity in nervous tissue. Using lead as a trapping agent (Wagner and Bitensky, 1974), several investigators have claimed to demonstrate adenylate cyclase activity in other mammalian tissues, such as liver cells (Reik et al., 1970), the islets of Langerhans (Howell and Whitfield, 1972) the capillary endothelium (Wagner et al., 1972) the nephron (Jande and Robert, 1974), and the uterine epithelium (Sananes and Psychoyos, 1974). Recently, however, serious criticism has been leveled at the use of the lead technique for the demonstration of adenylate cyclase * Supported by grants from the MedicalResearch Councilin the Academyof Finland
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activity (Lemay and Jarett, 1975). Lemay and Jarett (1975) using a biochemical method, have shown that lead, at the concentrations employed in the above studies, totally inhibits adenylate cyclase activity. The present paper describes a method in which cobalt is used as a precipitating agent for demonstrating adenylate cyclase activity in nervous tissue. Cobalt salts are visible in the electron microscope, and, moreover, Co + + was found to stimulate adenylate cyclase activity.
Material and Methods Adult male albino rats of Sprague-Dawley strain weighing about 250 g were used for the study. The experimental rats were injected with L-dopa (L-3,4-dihydroxyphenylalanine, L-dopa. Orion Oy, Finland) 100 nag kg/body wt. intraperitoneally daily during one week, and then killed with a sharp blow on the neck. The controls were given physiological saline. The anterior parts of the cortex and eaudate nuclei of the brain were quickly removed on chilled ice, and cut into thin slices with a razor blade or a tissue chopper (The Mickle Laboratory Engineering Co, England). The slices of brain cortex to be used for histochemical demonstration were pre-incubated at 37~ C for 20 rain in a medium (modified Krebs-Ringer-glucose solution) (Larrabee, 1958) buffered with Tris (hydroxymethyl) methyl-glycine (TRICINE, Sigma, St. Louis, Mo.) containing 1-3 retool/1 adenine (E. Merck AG, Darmstadt), or 1 3 mmol/1 adenosine (E. Merck AG, Darmstadt) together with 0.1 retool/1 norepinephrine hydrochloride (Fluka AG, Buchs SG). For the slices of caudate nuclei, the same medium was used, with the addition of 3 mmol/1 dopamine (Dopamin-hydrochlorid, Fluka AG, Buchs SG). The slices were then fixed in freshly prepared 1% paraformaldehyde-KrebsRinger-glucose (Larrabee, 1958) buffered with TRICINE to pH 7.0. After fixation for 5 min at 4~ C the slices were washed for 30 min in three changes of Krebs-Ringer-glucose solution. Some of the cortex slices were processed further without any prefixation. Histochemical incubation was carried out for 30 min at room temperature (22~ C) in a medium with the final concentration: TRICINE buffer (pH 8.3) 50 retool/l, MgCI2 3 mmol/1, CaC1/8 mmol/1, Co (NO3)2 3 retool/l, ATP disodium salt (Fluka AG, Bucks SG) 3 mmol/1, ouabain 1 mmol/I, theophylline 10 retool/l, and L-tetramisole (R 12564, Janssen Pharmaceutica, Netherlands) 0.5 mmol/ 1. The pH of the solution was adjusted to 8.3. The solution was clear and light pinkish in color. After incubation the slices were washed for 30 min in three changes of ice-cold physiological saline, post-fixed in 1% OsO4 for 1 h, dehydrated, and embedded in Epon-Aratdite mixture. The ultrathin sections were cut with diamond knives and viewed and photographed unstained with a Philips EM 300, at 40 kV. In preliminary studies the concentrations of all the reagents were varied over a wide range. In the control studies cobalt, ATP and inhibitors were omitted. ATP was replaced with/3-glycerophosphate (E. Merck AG, Darmstadt). The slices were heated at 100~ C for 10 min. In the preliminary studies the incubation media also included 2 retool/1 dithiotreitol or 10 retool/1 NaF, but because these compounds formed visible precipitates in the incubation solutions their use was abondoned. In some incubation solutions 5 retool/1 alloxan hydrate (Fluka AG, Buchs) was used. Some rats were given injections of haloperidol (Orion, Helsinki) 10 mg/kg/body wt. intraperitoneally 24 h before they were killed. The biochemical control assays of adenylate cyclase activity were performed with homogenates of cortical slices. The enzyme activity was measured after stimulation with adenosine-norepinephrine and the value compared with that for unstimulated control slices. Adenosine triphosphatase activity was checked from the cortical slices under the incubation conditions using the method by H/irk6nen et al. (1972). No biochemical activity was detected. Comparative values of adenylate cyclase activity were also measured after fixation for 5 min in 1% paraformaldehyde solution; likewise, the effect of 3 mmol/1 Co (NO3)2 in the Krebs-Ringer-glucose was tested. For details of the method used for measuring the activity of the enzyme see Hfirk6nen et al. (1974).
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Fig. 1. Dopamine-stimulated adenylate cyclase activity in the caudate nucleus of the rat. The reaction product is found in the plasma membrane of the large nerve fiber. Two terminal axons with synaptic vesicles are seen and the postsynaptic membrane exhibits enzyme activity (arrows). The neighboring nerve fibers show no visible enzyme activity. The b a r = 0 . 5 gm in all figures, x 13,200
Fig. 2. Norepinephrine-stimulated adenylate cyclase activity in the cerebral cortex of rat brain. The reaction product is seen at the postsynaptic site in the postsynaptic membrane (arrow). The myelinated nerve fiber and mitochondria (34) are negative, x 19,800
116
L. Rechardt and M. H/irk6nen Fig. 3. Dopamine-stimulated adenylate cyciase activity in the caudate nucleus of the rat. The reaction product seen in the postsynaptic membrane is identical to that seen in the cerebral cortex. The pre-synaptic part and the synaptic vesicles are devoid of any reaction products, x 13,200 Fig. 4. A control section of the cerebral cortex without any preincubation with amines. No reaction product due to adenosine triphosphatase activity is observed in mitochondria or in myelinated nerve fibers, which are the common localization sites of adenosine triphosphatase. • 13,200 Fig. 5. A control section of the cerebral cortex. Five mmol/1 alloxan hydrate was included in the incubation medium. Alloxan hydrate totally inhibited the enzymatic reaction in the slices, x 13,200
Observations F i x a t i o n for 5 m i n in 1% p a r a f o r m a l d e h y d e s o l u t i o n gave g o o d p r e s e r v a t i o n o f the overall u l t r a s t r u c t u r e o f b r a i n tissue, a n d especially o f the s y n a p t i c structures, including the s y n a p t i c vesicles. M i t o c h o n d r i a were s o m e t i m e s swollen a n d extracellular spaces widened. In sections e x a m i n e d w i t h o u t a n y prefixation, the nerve fibers, synapses a n d m i t o c h o n d r i a were s o m e w h a t inferior to I those in fixed slices b u t still identifiable. Brief fixation with 1% p a r a f o r m a l d e h y d e was preferred, however, to m a k e h a n d l i n g o f the tissue slices easier. Biochemically, this fixation p r o c e d u r e d i m i n i s h e d the s t i m u l a t e d a d e n y l a t e cyclase activity only 9 % as c o m p a r e d with the activity o f unfixed cortical slices otherwise t r e a t e d identically. W i t h o u t a d e n o s i n e - n o r e p i n e p h r i n e p r e i n c u b a t i o n , which i n c r e a s e d the enz y m e activity b y 23%, the scanty, r a n d o m l y d i s t r i b u t e d p r e c i p i t a t e ( p r o b a b l y
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due to the basal activity of adenylate cyclase) was not found in any specific structures (Fig. 4). It is certain that the precipitate formed did not represent adenosine triphosphatase activity, because the mitochondria and myelinated nerve fibres were devoid of the reaction product (Fig. 4). This was also confirmed biochemically. The reaction product which was easily visible in the electron microscope, was localized around some nerve fibers in both the cerebral cortex and the caudate nucleus (see Fig. 1). Two nerve terminals seem to synapse on this nerve fiber and the reaction product is seen continuously at the synaptic sites. In some of the synapses the reaction product was attached contiguous to the postsynaptic membrane (Fig. 2 and 3). The presynaptic site did not show any activity. The morphology, distribution and number of synapses were not recorded in detail in this connection, but will be restudied in the future. When 5 mmol/1 alloxan hydrate was added to the incubation medium no precipitate was seen (Fig. 5). Similarly, in the caudate nucleus pretreatment with haloperidol reduced the number of synapses showing the reaction product. However, the enzyme activity was not as completely inhibited as would be expected on the basis of biochemical studies (Kebabian et al., 1972). When ATP was replaced with/%glycerophosphate no reaction product was seen with either tissue. The other adequate histochemical controls were also negative.
Discussion
Severe criticism has been leveled at the use of lead as a trapping agent for demonstrating phosphatases (Rosenthal et al., 1966) and adenylate cyclase (Lemay and Jarett, 1975). Glutaraldehyde fixation, used earlier for the histochemical demonstration of adenylate cyclase activity, has likewise been shown to destroy most of this activity (Wagner and Bitensky, 1974). In the present study, therefore, we used neither lead nor glutaraldehyde and made biochemical tests for any possible false-positive reactions due to the presence of phosphatases of various kinds. We therefore decided to use cobalt as a trapping agent. In biochemical tests we found that cobalt enhances adenylates cyclase activity by 16%. In alkaline solutions cobalt rapidly forms a hydroxylic precipitate with pyrophosphate. The cobalt salt is well visualized in the electron microscope, especially at low voltages, such as 40 kV. The most serious problem in demonstrating adenylate cyclase activity with this agent is the false-positive reactions due to phosphatases. The enzymes produce inorganic phosphate, which is also precipitated by cobalt. Sodium fluoride cannot be used to inhibit phosphatase, because it, too, forms a precipitate. We therefore tested several other ways to overcome this difficulty. Adenylate cyclase is active over a relatively broad pH range (von Hungen and Roberts, 1974; Clement-Cormier et al., 1975), and in the present study the upper limit of the pH range was adopted. At this pH the activities of adenosine phosphatases and nucleotidases are depressed, and in the incubation conditions used no adenosine triphosphatase activity was detectable biochemically in the brain slices.
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Even the mitochondria were as a rule negative. As a substrate, 5'-adenylylimidodiphosphate has been claimed to be a more specific for adenylate cyclase than ATP (Wagner and Bitensky, 1974). So far we have not used it, because in studies on localization with the lead techniques generally the two substrates gave identical results (see Wagner and Bitensky, 1974). Theophylline at a concentration as high as was used here inhibits phosphodiesterase activity (Cheung, 1970), which theoretically could interfere with the present method. Theophylline also prevents the further formation of monophosphates in the enzymatic cycle. Alloxan hydrate does not inhibit phosphodiesterase activity (Cohen and Bitensky, 1969), and as the alloxan controls were n~gative, it seems unlikely that phosphodiesterase was responsible for the activity demonstrated in the synapses by the present method. The other serious source of error might be the heterogeneous group of alkaline phosphatases, which could cause precipitates with ATP as substrate at pH 8.3. The introduction of a specific inhibitor of alkaline phosphatases by Borgers (1973) certainly has simplified the demonstration of phosphatases. In the present study no activity was observed with /?-glycerophosphate as substrate. Alloxan, moreover, does not inhibit either adenosine or alkaline phosphatases (Cohen and Bitensky, 1969) ; thus it presumably inhibited only the adenylate cyclase activities localized by the present technique. The paraformaldehyde fixation made it impossible to demonstrate the small dense-cored vesicles connected with aminergic transmission. The synapses contained only clear vesicles. It has been suggested that the ultrastructural localization of adenylate cyclase activity would demonstrate synapses containing norepinephrine receptors in the cerebral cortex and dopamine receptors in the caudate nucleus (Greengard et al., 1972). The postsynaptic histochemical localization observed in the present study confirms the results of biochemical measurements after pharmacological and denervation experiments, which showed that the adenylate cyclase connected with nervous transmission is located postsynaptically (Greengard et al., 1972). So far, no distinct presynaptic location has been observed in the brain tissue of the rat, although recently it has been proposed that adenylate cyclase is involved presynaptically in the synthesis of tyrosine hydroxylase and in microtubular function (Greengard, 1975). However, in cultured sympathetic ganglia of the chicken we have observed a presynaptic localization in the nerve fibers (Hervonen and Rechardt, in press), a finding which would confirm the hypothesis proposed by Greengard (1975). References Borgers, M.: The cytochemical application of new potent inhibitors of alkaline phosphatases. J. Histochem. Cytochem. 21, 812-824 (1973) Cheung, W.Y.: Cyclic nucleotide phosphodiesterase. In: Role of cyclic A M P in cell function (P. Greengard., E. Costa, ed.), Vol. 3, pp. 51 65. New York: Raven Press 1970 Clement-Cormier, Y.C., Parrish, R.G., Petzold, G.L., Kebabian, J.W., Greengard, P. : Characterization of a dopamine-sensitive adenylate cyclase in the rat caudate nucleus. J. Neurochem. 25, 143 149 (1975) Cohen, K.L., Bitensky, M.W. : Inhibitory effects of alloxan on mammalian adenyl cyclase. J. Pharmacol. exp. Ther. 169, 80-86 (1969)
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Greengard, P.: Prexynaptic and postsynaptic roles of cyclic AMP and protein phosphorylation at catecholaminergic synapses. In: Chemical tools in catecholamine research (O. Almgren., A. Carlsson., J. Engel, ed.), Vol. 2, pp. 249 256. Amsterdam: North-Holland Publishing Company 1975 Greengard, P., McAfee, D.A., Kebabian, J.W.: On the mechanism of action of cyclic AMP and its role in synaptic transmission. In: Advances in cyclic nucleotide research, Vol. 1, pp. 337 355. New York: Raven Press 1972 H~irkSnen, M., Hopsu-Havu, V.K., Raij, K.: Cyclic adenosine monophosphate, adenyl cyclase and cyclic nucleotide phosphodiesterase in psoriatic epidermis. Acta derm.-venerol. (Stockh.) 54, 13-18 (1974) Hfirk6nen, M., Palkama, A., Uusitalo, R. : Functional dependence of the ciliary epithelium ATPase activity and intraocular pressure on the autonomic nervous system. Acta physiol, scand. 86, 327 341 (1972) Hervonen, H., Rechardt, L. : Histochemical localization of adenylate cyclase in cultured sympathetic neurons. Histochemistry 48, 43-50 (1976) Howell, S.L., Whitfield, M. : Cytochemical localization of adenyl cyclase activity in rat islets of Langerhans. J. Histochem. Cytochem. 20, 873-879 (1972) Huang, M., Shimizu, J., Daly, J.: Regulation of adenosine cyclic 3',5'-phosphate formation in cerebral cortical slices, Molec. Pharmacol. 7, 155-162 (1971) Hungen, K. von, Roberts, S.: Neurotransmitter-sensitive adenylate cyclase systems in the brain. In: Reviews of neuroscience (S. Ehrenpreis., I. Kopin, ed.), Vol. l, pp. 231-281. New York: Raven Press 1974 Jande, S.S., Robert, P. : Cytochemical localization of parathyroid hormone activated adenyl cyclase in rat kidney. Histochemistry 40, 323-327 (1974) Kebabian, J.W., Petzold, G.L., Greengard, P.: Dopamine-sensitive adenylate cyclase in caudate nucleus of rat brain, and its similarity for the "dopamine receptor". Proc. nat. Acad. Sci. (Wash.) 69, 2145-2149 (1972) Larrabee, M.G.: Oxygen consumption of excised sympathetic ganglia at rest and in activity. J. Neurochem. 2, 81 10t (1958) Lemay, A., Jarett, L.: Pitfalls in the use of lead nitrate for the histochemical demonstration of adenylate cyclase activity. J. Ceil Biol. 65, 39-50 (1975) Perkins, J.P.: Adenyl cyclase. In: Advances in cyclic nucleotide research, Vol. 3, pp. 1~54. New York: Raven Press 1973 Reik, L., Petzold, G.L., Higgins, J.A., Greengard, P., Barrnett, R.J.: Hormone sensitive adenyl cyclase: cytochemical localization in the rat liver. Science 168, 382-384 (1970) DeRobertis, E., Arnaiz, G.R.D.L., Alberici, M., Butcher, R.W., Sutherland, E.W.: Subcellular distribution of adenyl cyclase and cyclic phosphodiesterase in rat brain cortex. J. biol. Chem. 242, 3487 3493 (1967) Rosenthal, A.S., Moses, H.L., Beaver, D.L., Schuffman, S.S. : Lead ion and phosphatase histochemistry. I. Non-enzymatic hydrolysis of nucleoside phosphates by lead ion. J. Histochem. Cytochem. 14, 698-701 (1966) Sananes, N., Psychoyos, A. : Cytochemical localization of adenyl cyclase in the rat uterus. J. Repr Fertil. 38, 181-183 (1974) Wagner, R.C., Bitensky, M.W. : Adenylate cyclase in electron microscopy of enzymes. In: Electt, microscopy of enzymes (M.A. Hayat, ed.), pp. 110-131. New York: Van Norstrand Company Inc. 1974 Wagner, R.C., Kreiner, P., Barrnett, R.J., Bitensky, M.W. : Biochemical characterization and cytochemical localization of a catecholamine-sensitive adenylate cyclase in isolated capillary endothelium. Proc. nat. Acad. Sci. (Wash.) 69, 3176-3179 (1972)
Received October 10, 1976
