Journal of Nrurochemisrry Vol. 33. pp. I165 to I168 Pergarnon Press Ltd 1979. Printed in Great Britain 0 International Society lor Neurochernistry Ltd
0022-3042/79/1201-1165102.00/0
MUSCARINIC CHOLINERGIC STIMULATION INCREASES CYCLIC GMP LEVELS IN RAT HIPPOCAMPUS' ASA C. BLACK,JR., DEANSANDQUIST, JAMESR. WEST, JAMESK. WAMSLEY' and TERENCE H. WILLIAMS Department of Anatomy, College of Medicine, University of Iowa, Iowa City, IA 52242, U.S.A. (Received 5 March 1979. Accepted 4 May 1979)
Abstract-Muscarinic cholinergic agonists increase cyclic G M P levels in a number of neural tissues. Since the rat hippocampus receives a cholinergic innervation from the septum, we decided to test whether cyclic GMP levels of the rat hippocampus are increased by bethanechol, a muscarinic cholinergic agonist. Incubation of rat hippocampi with varying concentrations of bethanechol showed that the increase in cyclic GMP levels is concentration-dependent, 500 pwbethanechol producing a maximum increase of 490% over control values. The bethanechol-evoked increases were blocked by the muscarinic antagonist atropine, and were calcium-dependent. It is concluded that at least some of the cells projecting to the rat hippocampus form muscarinic cholinergic synapses which act via a cyclic GMP-dependent mechanism.
EVIDENCE is accumulating to support the view that MATERIALS AND METHODS guanosine 3',5'-monophosphate (cyclic GMP) is inAdult Sprague-Dawley rats of either sex were killed by volved in neural transmission (GREENGARD, 1978). decapitation and the entire brain was removed quickly. Muscarinic cholinergic agonists have been shown to Hippocampi were isolated by blunt dissection and placed increase cyclic G M P levels in rabbit cerebral cortex in ice-cold Eagle's Medium (Grand Island Biological Co., (LEEet al., 1972) and cerebellum (Kuo et al., 1972), Catalog No. 138). Whole hippocampi were pre-incubated rat cerebral cortex (PALMER& DUSZYNSKI, 197.51, and for 30min at 37°C in Eagle's Medium containing 5 mMthe superior cervical ganglia of both cow (KEBABIANtheophylline, using a shaking incubator open to the atmoset a/., 19756) and guinea pig (WAMSLEY et al., 1979). phere. They were then incubated for varying periods in The effect of muscarinic agonists on cyclic G M P the same solution containing 2.2 mM-CaC1, and the approlevels is calcium-dependent and is blocked by atro- priate concentration of bethanechol (Sigma Chemical Co.). Upon completion of incubation, samples were frozen impine. Depolarization by 1 0 0 p ~ - K + mimics the effect mediately and stored in liquid nitrogen until assay. They of muscarinic cholinergic stimulation in bovine super- were homogenized in 2 ml of ice-cold 6% trichloroacetic ior cervical ganglia (BARTFAI et al., 1978). acid using a Polytron tissue disintegrator and centrifuged Reports indicate that the rat hippocampus receives at 12,000g for 30 min at 4°C in a Sorvall RC-5. Precipia cholinergic innervation which is derived from cell tates were dissolved in 1.0 M-NaOH and analyzed for probodies located in the medial septa1 nucleus, the nu- tein by the method of WRY et a / . (1951) using bovine cleus of the diagonal band and possibly the interme- serum albumin as standard. Supernatants were extracted diolateral regions of the septum (LEWIS& SHUTE, 5 times with twice their volume of diethyl ether saturated 1967; MEIBACH& SIEGEL,1977; MELLGREN & SRE- with double-distilled water. Traces of ether were removed BRO,1973). These afferents are thought to synapse on by placing the samples in a water bath at 40°C for a few minutes. Samples were then frozen, lyophilized, resuspyramidal cells of the hippocampus (KUHAR & YAMA- pended in 0.05 M-sodium acetate buffer (pH 6.2) and MURA, 1975). Moreover, hippocampal pyramidal cells assayed for cyclic GMP according to the method of can be stimulated by acetylcholine applied by ionto- STEINER(1974), using radioimmunoassay materials purphoresis (BISCOE & STRAUGHAN, 1966). The presence chased from New England Nuclear Corp. Results are of a cholinergic innervation to this important area reported as pmol cyclic GMP S.E.M. of the rat brain led us to study the effect of muscarinic cholinergic stimulation on cyclic G M P levels in rat RESULTS hippocampus. Incubation of rat hippocampi with 500 pM-bethane'Supported in part by NS-11650 to T.H.W. and CA chol produced a time-dependent increase in cyclic 2424142 to D.S. from the National Institutes of Health, GMP levels (Fig. 1). Incubation of rat hippocampi and by National Research Service Award PHS for 2.5min with varying concentrations of bethanelT-32MH15172Zol to J.K.W. N.J.M.H. Predoctoral Fellow. Present address: Depart- chol produced a concentration-dependent increase in ment of Pharmacology and Experimental Therapeutics, cyclic GMP levels (Fig. 2). Five hundred micromolar The Johns Hopkins School of Medicine, 725 North Wolfe bethanechol produced the greatest increase (490% Street, Baltimore, MD 21205, U.S.A. over control .values). The increases produced by beth1165
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FIG.I. Time course for the effect of bethanechol on cyclic GMP levels, using whole rat hippocampi. Hippocampi were pre-incubated for 30 rnin in Eagle’s Medium containing 5 mht-theophylline at 37°C. They were then incubated in the same solution which had been made 500 pht in bethanechol and 2.2 mM in CaCI,. Results are reported as pmol cyclic G M P per mg protein S.E.M. Each value represents the mean of 4-8 samples (number in parentheses). For all values, P < 0.001 (Student’s t-test).
anechol were blocked by the muscarinic antagonist atropine and were calcium-dependent (Fig. 3). DISCUSSION
A role for cyclic GMP in neuronal function is now well-documented (GREENGARD, 1978; GREENGARD & KEBABIAN, 1974; KEBABIAN, 1977; NATHANSON, 1977). There is no evidence that supports this view better than the studies of the superior cervical ganglion. KEBABIAN et al. (1975b) showed that the muscarinic
I 100
Beth +A t r
Beth Control -Ca++
FIG.3. Effect of atropine and calcium on the elevations of cyclic GMP levels produced by bethanechol in whole rat hippocampi. Samples labeled ‘Beth’ were pre-incubated in Eagle’s Medium containing 5 mht-theophylline for 30min at 37°C. They were then incubated in Eagle’s Medium containing 500 jmbethanechol, 2.2 mht-CaCI, and 5 mht-theophylline for 2.5 rnin at 37°C. Samples labeled ‘Beth + Atr’ were pre-incubated for 25 rnin in Eagle’s Medium containing 5 mht-theophylline and then for 15 min in the same solution containing 300 pht-atropine sulfate. They were then incubated in Eagle’s Medium containing 500 pht-bethanechol, 2.2 mht-CaCI, and 5 mM-theophylline for 2.5min at 37°C. Samples labeled ‘Beth - Ca2” were treated in the same manner as the ‘Beth’ samples except that Ca2+ was omitted from the incubation medium. Control samples were pre-incubated in Eagle’s Medium containing 5 mM-theophylline for 30 min, then incubated in the same solution containing 2.2mM-CaC1, for 2.5min. Results are reported as pmol cyclic G M P per mg protein k S.E.M. Each value represents the mean of 4-8 samples (number in parentheses). For Beth, P < 0.001 (Student’s t-test).
I
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Fic. 2. Effect of varying concentrations of bethanechol on cyclic GMP levels of whole rat hippocampi. Hippocampi were pre-incubated for 30 min in Eagle’s Medium containing 5-mht-theophylline at 37°C. They were then incubated for 2.5 rnin at 37°C in the same solution containing 2.2 mht-CaCI, and various concentrations of bethanechol. Results are reported as pmol cyclic GMP per mg protein k S.E.M. Each value represents the mean of 4 6 samples (number in parentheses). For all values, P < 0.001 (Student’s t-test). The control value is 0.091 k 0.02 pmol cyclic GMP per mg protein.
Cyclic GMP and the rat hippocampus
1167
1970). BISCOE& STRAUGHAN cholinergic agonist bethanechol elevates cyclic G M P 1970; STORM-MATHISEN, levels in bovine SCG; an effect that was blocked by (1966) have shown that hippocampal pyramidal cells atropine (a muscarinic cholinergic antagonist) but not can be activated by the iontophoretic application of by hexamethonium (a nicotinic cholinergic antagon- acetylcholine, and this excitation is selectively blocked ist). KEBABIAN et al. (1975a) demonstrated by an im- by atropine. More recently, KUHAR& YAMAMURA munocytochemical technique that the increases in (1975) used autoradiographic localization of tritiated cyclic G M P in the bovine superior cervical ganglion quinuclidin yl benzilate to localize muscarinic chofollowing incubation in acetylcholine occur in princi- linergic receptors in the rat hippocampus. They found pal ganglionic neurons. Either preganglionic physio- the highest densities of silver grains over the stratum logical or muscarinic cholinergic pharmacological oriens, stratum radiatum and stratum moleculare. stimulation increases cyclic G M P levels in guinea pig Taken together, these studies suggest that the septum gives rise to a cholinergic projection to the basal (straSCG (WAMSLEY et al., 1979). The effects of muscarinic agonists are calcium-dependent (BARTFAI et al., 1978; tum oriens) and apical (stratum radiatum) dendrites of the hippocampal pyramidal cells. GREENGARD, 1978). Recently, CLEMENT-CORMIER et a/. (1978) reported Investigators who have considered the matter believe that, in the ganglion, acetylcholine released that acetylcholine and cyclic G M P each had an exfrom preganglionic terminals interacts with muscar- citatory effect upon rabbit hippocampal pyramidal inic cholinergic receptors, causing increased cyclic cell responses to electrophysiological stimulation of G M P levels in principal ganglionic neurons (GREEN- the pathway from the medial septal region to hippoCARD, 1978; GREENCARD & KEBABIAN, 1974; KEBA- campal field CAI. The phosphodiesterase inhibitor isobutylmethylxanthine prolonged the time course of BIAN, 1977). Exogenous cyclic G M P and its derivatives mimic the depolarizing action of acetylcholine recovery of responses to post-tetanic stimulation. on principal ganglionic neurons of the superior cervi- Electrical stimulation of the medial septal region procal ganglion (MCAFEE& GREENGARD, 1972; DUN et duced a 2-fold increase in hippocampal cyclic G M P al., 1977a, b, 1978; GALLAGHER& SHINNICK-GAL-levels in the rabbit. It seems reasonable to infer that cyclic G M P is LAGHER,1977; HASHIGUCHI et al., 1978). ECCLES& LIBET(1961) postulated that a muscarinic cholinergic generated by activity in the cholinergic projection receptor on the principal ganglionic neuron is in- from the septum to the hippocampus in the rat. The volved in induction of the slow excitatory postsynap- cyclic G M P increases seen following stimulation pretic potential (s-EPSP) seen following preganglionic sumably occur in the pyramidal cells, although this stimulation in rabbit superior cervical ganglia. The has not been confirmed by immunohistochemistry. s-EPSP produced by preganglionic physiological Our results and those of KUHAR& YAMAMURA (1975) stimulation and blocked by atropine can be mimicked indicate that the synapses between the cholinergic by exogenous 50 PM-dibutyryl cyclic G M P (MCAFEE fibers and the pyramidal cells possess muscarinic cho& GREENGARD, 1972). LIBEThas shown that dopa- linergic receptors. Thus in the central nervous system, mine causes a marked potentiation and prolongation as in the peripheral nervous system, cyclic G M P of the s-EPSP induced in rabbit superior cervical apparently plays a role in synaptic transmission in ganglia by preganglionic stimulation. Cyclic AMP muscarinic cholinergic pathways. may have a role in potentiation of the s-EPSP (LIBET et al., 1975; KOBAYASHI et al., 1978). REFERENCES In the central nervous system, cyclic G M P causes R. E. & GREENGARD P. (1978) Muscarexcitation of the pyramidal tract neurons in rat cere- BARTFAIT., STUDY inic stimulation and cGMP synthesis in the nervous sysbral cortex (STONEet al., 1975) and mimics the effects of acetylcholine on cat neocortical neurons (SWARTZ tem, in Adoances in Behavioral Biology, Volume 24: Cholinergic Mechanisms and Psychopharmacology (JENDEN & WOODY, 1976). D. E., ed.) pp. 285-295. Plenum Press, New York. The rat hippocampus receives a cholinergic septal BISCOET. J. & STRAUGHAN D. W. (1966) Micro-electroinput derived from cell bodies located in the medial phoretic studies of neurones in the cat hippocampus. septal nucleus, the nucleus of the diagonal band, and J. Physiol., Lond. 183, 341-359. possibly the intermediolateral regions of the septum CLEMENT-CORMIER Y., DIVAKARAN P., DE FRANCE J., STANLEY J. & MARCHAND J. (1978) Elevation of cyclic GMP (LEWIS & SHUTE, 1967; MEIBACH& SIEGEL, 1977; levels in the hippocampus following electrical stimuMELLGREN & SREBRO,1973). Anatomically, the septolation of the medial septal region. Fedn Proc. Fedn Am. hippocampal projection is one of the best-studied SOCSexp. Biol. 37, 524 (Abstr. No. 1634). cholinergic pathways in the central nervous system. K. & KARCZMAR A. G. (1977a) DopaElectron microscopic studies indicate that most ace- DUNN. J.. KAIBARA mine and adenosine 3’,5’-monophosphateresponses of tylcholinesterase-positive terminals in the hipposingle mammalian sympathetic neurons. Science 197, campus form axodendritic synapses (SHUTE& LEWIS, 778-780. 1966). Detailed histochemical studies indicate that the DUNN. J., KAIBARA K. & KARCZMAR A. G. (1977b) Direct highest levels of hippocampal acetylcholinesterase postsynaptic membrane effect of dibutyryl cyclic GMP and choline acetyltransferase are located in the straon mammalian sympathetic neurons. Neuropharmacotum oriens, stratum radiatum and the hilus (FONNUM, logy 16, 7 1 ~ 7 1 7 .
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3’:5’-cyclic monophosphate content in mammalian DUNN. J., KAIBARA K. & KARCZMAR A. G. (1978) Muscarbrain, heart muscle and intestinal smooth muscle. Proc. inic and cGMP induced membrane potential changes: natn. Acad. Sci., U.S.A. 69, 3287-3291. differences in electrogenic mechanisms. Brain Res. 150, LEWISP. R. & SHUTEC. C. D. (1967) The cholinergic lim658-66 1. ECCLESR. M. & LIBETB. (1961) Origin and blockade of bic system: projections to the hippocampal formation, medial cortex, nuclei of the ascending cholinergic reticuthe sympathetic responses of curarized sympathetic lar system, and the subfornical organ and supraoptic ganglia. J . Physiol., Lond. 157, 484-503. cortex. Brain 90, 521-540. FONNUM F. (1970) Topographical and subcellular localizaH. & TANAKA T. (1975) Synaptic LIBETB., KOBAYASHI tion of choline acetyltransferase in the rat hippocampal coupling in the production and storage of a neuronal region. J . N eurcchem. 17, 1029- 1037. memory trace. Nature, Lond. 258, 155-1 57. P. (1977) ElecGALLAGHER J. P. & SHINNICK-GALLAGHER 0. H., ROSEBROUGHN. J., FARR A. L. & RANDALL trophysiological studies of cyclic nucleotides injected in- LOWRY R. J. (1951) Protein measurement with the Fohn phenol tracellularly into rat superior cervical ganglion cells. reagent. J . biol. Chem. 193, 265-275. Science 198, 851-852. MCAFEE D. A. & GREENGARD P. (1972) Adenosine GREENCARD P. (1978) Cyclic Nucleotides, Phosphorylated Proteins and Neuronal Function. Raven Press, New 3’,5’-monophosphate: electrophysiological evidence for a York. role in synaptic transmission. Science 178, 3 1&3 12. J. W. (1974) Role of cyclic MEIBACHR. C. & SIEGELA. (1977) Efferent connections GREENGARD P. & KEBABIAN of the septal area in the rat: an analysis utilizing retroAMP in synaptic transmission in the mammalian perigrade and anterograde transport methods. Brairi Res. pheral nervous system. Fedn Proc. Fedn Am. SOCSexp. Biol. 33, 1059-1067. 119, 1-20. T., USHIYAMA N. S., KOBAYASHI H. & LIBET MELLGREN HASHIGUCHI S. I. & SREBRO B. (1973) Changes in acetylchoB. (1978) Does cyclic G M P mediate the slow excitatory linesterase and distribution of degenerating fibers in the hippocampal region after septal lesions in the rat. Brain postsynaptic potential in sympathetic ganglia? Nature. Lond. 271, 267-268. Res. 52, 19-36. KEBABIAN J. W. (1977) Biochemical regulation and physio- NATHANSON J. A. (1977) Cyclic nucleotides and nervous logical significance of cyclic nucleotides in the nervous system function. Physiol. Rev. 57, 157-256. system, in Advances in Cyclic Nucleoride Research C. (1975) Regional cyclic PALMER G. C. & DUSZYNSKI (GREENGARD P. & ROBISONG. A., eds.) Vol. 8, pp. G MP content in incubated tissue slices of rat brain. Eur. 421-507. Raven Press, New York. J . Pharmac. 32, 375-379. J. W., BLOOM F. E.. STEINER A. L. & GREENGARDSHUTEC. C. D. & LEWISP. R. (1966) Electron microscopy KEBABIAN of cholinergic terminals and acetylcholinesterase-conP. (19754 Neurotransmitters increase cyclic nucleotides taining neurons in hippocampal formation of the rat. in postganglionic neurons: immunocytochemical demonZ . Zelffosch. 69,334343. stration. Science 190, 157-159. A. L. & GREENGARD P. (1975b) STEINERA. L. (1974) Assay of cyclic nucleotides by KEBABIAN J. W., STEINER Muscarinic cholinergic regulation of cyclic guanosine radioimmunoassay methods, in Methods in Enzymology, Volume 38. Hormone Action. Part C : Cyclic Nucleotides 3’,5’-monophosphate in autonomic ganglia: possible role (OMALLEY B. W. & HARDMAN J. G.. eds.) pp. 96105. in synaptic transmission. J . Pharmac. exp. Ther. 193, 474488. Academic Press, New York. T. & USHIYAMA N. S . (1978) STONET. W., TAYLOR KOBAYASHI H., HASHIGUCHI D. A. & BLOOMF. E. (1975) Cyclic Postsynaptic modulation of excitatory process in symAMP and cyclic G MP may mediate opposite neuronal pathetic ganglia by cyclic AMP. Nature, Lond. 271, responses in the rat cerebral cortex. Science 187, 268-270. 845-847. KUHARM. J. & YAMAMURAH. (1975) Cholinergic muscar- STORM-MATHISEN J. (1970) Quantitative histochemistry of inic receptors in rat brain: light autoradiographic localacetylcholinesterase in rat hippocampal region correization by the specific binding of a potent antagonist. lated to histochemical staining. J . Neurochem. 17, Nature, Lond. 253, 560-561. 739-750. K. G., DONNELLY SWARTZB. & WOODYC. D. (1976) Correlated effects of Kuo J.-F., LEET. P., REYESP. L., WALTON P. (1972) Cyclic nucleotideT. E., JR. & GREENGARD acetylcholine and cyclic GMP on input resistance of dependent protein kinases. X. An assay method for neocortical neurons in awake cats. Soc. Nettrosci. Abstr. the measurement of guanosine 3’,5‘-monophosphate in 2, Part 2, 802. various biological materials and a study of agents regu- WAMSLEY J. K., WESTJ. R., BLACKA. C., JR. & WILLIAMS lating its levels in heart and brain. J . biol. Chem. 247, T. H. (1979) Muscarinic cholinergic and preganglionic 1622. physiological stimulation increase cyclic GMP levels in P. (1972) Role of musLEET. P., Kuo J.-F. & GREENGARD guinea pig superior cervical ganglion. J . Neurochem. 32, carinic cholinergic receptors in regulation of guanosine 1033-1 035.
0022-3042/79/1201-1165102.00/0
MUSCARINIC CHOLINERGIC STIMULATION INCREASES CYCLIC GMP LEVELS IN RAT HIPPOCAMPUS' ASA C. BLACK,JR., DEANSANDQUIST, JAMESR. WEST, JAMESK. WAMSLEY' and TERENCE H. WILLIAMS Department of Anatomy, College of Medicine, University of Iowa, Iowa City, IA 52242, U.S.A. (Received 5 March 1979. Accepted 4 May 1979)
Abstract-Muscarinic cholinergic agonists increase cyclic G M P levels in a number of neural tissues. Since the rat hippocampus receives a cholinergic innervation from the septum, we decided to test whether cyclic GMP levels of the rat hippocampus are increased by bethanechol, a muscarinic cholinergic agonist. Incubation of rat hippocampi with varying concentrations of bethanechol showed that the increase in cyclic GMP levels is concentration-dependent, 500 pwbethanechol producing a maximum increase of 490% over control values. The bethanechol-evoked increases were blocked by the muscarinic antagonist atropine, and were calcium-dependent. It is concluded that at least some of the cells projecting to the rat hippocampus form muscarinic cholinergic synapses which act via a cyclic GMP-dependent mechanism.
EVIDENCE is accumulating to support the view that MATERIALS AND METHODS guanosine 3',5'-monophosphate (cyclic GMP) is inAdult Sprague-Dawley rats of either sex were killed by volved in neural transmission (GREENGARD, 1978). decapitation and the entire brain was removed quickly. Muscarinic cholinergic agonists have been shown to Hippocampi were isolated by blunt dissection and placed increase cyclic G M P levels in rabbit cerebral cortex in ice-cold Eagle's Medium (Grand Island Biological Co., (LEEet al., 1972) and cerebellum (Kuo et al., 1972), Catalog No. 138). Whole hippocampi were pre-incubated rat cerebral cortex (PALMER& DUSZYNSKI, 197.51, and for 30min at 37°C in Eagle's Medium containing 5 mMthe superior cervical ganglia of both cow (KEBABIANtheophylline, using a shaking incubator open to the atmoset a/., 19756) and guinea pig (WAMSLEY et al., 1979). phere. They were then incubated for varying periods in The effect of muscarinic agonists on cyclic G M P the same solution containing 2.2 mM-CaC1, and the approlevels is calcium-dependent and is blocked by atro- priate concentration of bethanechol (Sigma Chemical Co.). Upon completion of incubation, samples were frozen impine. Depolarization by 1 0 0 p ~ - K + mimics the effect mediately and stored in liquid nitrogen until assay. They of muscarinic cholinergic stimulation in bovine super- were homogenized in 2 ml of ice-cold 6% trichloroacetic ior cervical ganglia (BARTFAI et al., 1978). acid using a Polytron tissue disintegrator and centrifuged Reports indicate that the rat hippocampus receives at 12,000g for 30 min at 4°C in a Sorvall RC-5. Precipia cholinergic innervation which is derived from cell tates were dissolved in 1.0 M-NaOH and analyzed for probodies located in the medial septa1 nucleus, the nu- tein by the method of WRY et a / . (1951) using bovine cleus of the diagonal band and possibly the interme- serum albumin as standard. Supernatants were extracted diolateral regions of the septum (LEWIS& SHUTE, 5 times with twice their volume of diethyl ether saturated 1967; MEIBACH& SIEGEL,1977; MELLGREN & SRE- with double-distilled water. Traces of ether were removed BRO,1973). These afferents are thought to synapse on by placing the samples in a water bath at 40°C for a few minutes. Samples were then frozen, lyophilized, resuspyramidal cells of the hippocampus (KUHAR & YAMA- pended in 0.05 M-sodium acetate buffer (pH 6.2) and MURA, 1975). Moreover, hippocampal pyramidal cells assayed for cyclic GMP according to the method of can be stimulated by acetylcholine applied by ionto- STEINER(1974), using radioimmunoassay materials purphoresis (BISCOE & STRAUGHAN, 1966). The presence chased from New England Nuclear Corp. Results are of a cholinergic innervation to this important area reported as pmol cyclic GMP S.E.M. of the rat brain led us to study the effect of muscarinic cholinergic stimulation on cyclic G M P levels in rat RESULTS hippocampus. Incubation of rat hippocampi with 500 pM-bethane'Supported in part by NS-11650 to T.H.W. and CA chol produced a time-dependent increase in cyclic 2424142 to D.S. from the National Institutes of Health, GMP levels (Fig. 1). Incubation of rat hippocampi and by National Research Service Award PHS for 2.5min with varying concentrations of bethanelT-32MH15172Zol to J.K.W. N.J.M.H. Predoctoral Fellow. Present address: Depart- chol produced a concentration-dependent increase in ment of Pharmacology and Experimental Therapeutics, cyclic GMP levels (Fig. 2). Five hundred micromolar The Johns Hopkins School of Medicine, 725 North Wolfe bethanechol produced the greatest increase (490% Street, Baltimore, MD 21205, U.S.A. over control .values). The increases produced by beth1165
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FIG.I. Time course for the effect of bethanechol on cyclic GMP levels, using whole rat hippocampi. Hippocampi were pre-incubated for 30 rnin in Eagle’s Medium containing 5 mht-theophylline at 37°C. They were then incubated in the same solution which had been made 500 pht in bethanechol and 2.2 mM in CaCI,. Results are reported as pmol cyclic G M P per mg protein S.E.M. Each value represents the mean of 4-8 samples (number in parentheses). For all values, P < 0.001 (Student’s t-test).
anechol were blocked by the muscarinic antagonist atropine and were calcium-dependent (Fig. 3). DISCUSSION
A role for cyclic GMP in neuronal function is now well-documented (GREENGARD, 1978; GREENGARD & KEBABIAN, 1974; KEBABIAN, 1977; NATHANSON, 1977). There is no evidence that supports this view better than the studies of the superior cervical ganglion. KEBABIAN et al. (1975b) showed that the muscarinic
I 100
Beth +A t r
Beth Control -Ca++
FIG.3. Effect of atropine and calcium on the elevations of cyclic GMP levels produced by bethanechol in whole rat hippocampi. Samples labeled ‘Beth’ were pre-incubated in Eagle’s Medium containing 5 mht-theophylline for 30min at 37°C. They were then incubated in Eagle’s Medium containing 500 jmbethanechol, 2.2 mht-CaCI, and 5 mht-theophylline for 2.5 rnin at 37°C. Samples labeled ‘Beth + Atr’ were pre-incubated for 25 rnin in Eagle’s Medium containing 5 mht-theophylline and then for 15 min in the same solution containing 300 pht-atropine sulfate. They were then incubated in Eagle’s Medium containing 500 pht-bethanechol, 2.2 mht-CaCI, and 5 mM-theophylline for 2.5min at 37°C. Samples labeled ‘Beth - Ca2” were treated in the same manner as the ‘Beth’ samples except that Ca2+ was omitted from the incubation medium. Control samples were pre-incubated in Eagle’s Medium containing 5 mM-theophylline for 30 min, then incubated in the same solution containing 2.2mM-CaC1, for 2.5min. Results are reported as pmol cyclic G M P per mg protein k S.E.M. Each value represents the mean of 4-8 samples (number in parentheses). For Beth, P < 0.001 (Student’s t-test).
I
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250
500
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Bethanechol ( p M )
Fic. 2. Effect of varying concentrations of bethanechol on cyclic GMP levels of whole rat hippocampi. Hippocampi were pre-incubated for 30 min in Eagle’s Medium containing 5-mht-theophylline at 37°C. They were then incubated for 2.5 rnin at 37°C in the same solution containing 2.2 mht-CaCI, and various concentrations of bethanechol. Results are reported as pmol cyclic GMP per mg protein k S.E.M. Each value represents the mean of 4 6 samples (number in parentheses). For all values, P < 0.001 (Student’s t-test). The control value is 0.091 k 0.02 pmol cyclic GMP per mg protein.
Cyclic GMP and the rat hippocampus
1167
1970). BISCOE& STRAUGHAN cholinergic agonist bethanechol elevates cyclic G M P 1970; STORM-MATHISEN, levels in bovine SCG; an effect that was blocked by (1966) have shown that hippocampal pyramidal cells atropine (a muscarinic cholinergic antagonist) but not can be activated by the iontophoretic application of by hexamethonium (a nicotinic cholinergic antagon- acetylcholine, and this excitation is selectively blocked ist). KEBABIAN et al. (1975a) demonstrated by an im- by atropine. More recently, KUHAR& YAMAMURA munocytochemical technique that the increases in (1975) used autoradiographic localization of tritiated cyclic G M P in the bovine superior cervical ganglion quinuclidin yl benzilate to localize muscarinic chofollowing incubation in acetylcholine occur in princi- linergic receptors in the rat hippocampus. They found pal ganglionic neurons. Either preganglionic physio- the highest densities of silver grains over the stratum logical or muscarinic cholinergic pharmacological oriens, stratum radiatum and stratum moleculare. stimulation increases cyclic G M P levels in guinea pig Taken together, these studies suggest that the septum gives rise to a cholinergic projection to the basal (straSCG (WAMSLEY et al., 1979). The effects of muscarinic agonists are calcium-dependent (BARTFAI et al., 1978; tum oriens) and apical (stratum radiatum) dendrites of the hippocampal pyramidal cells. GREENGARD, 1978). Recently, CLEMENT-CORMIER et a/. (1978) reported Investigators who have considered the matter believe that, in the ganglion, acetylcholine released that acetylcholine and cyclic G M P each had an exfrom preganglionic terminals interacts with muscar- citatory effect upon rabbit hippocampal pyramidal inic cholinergic receptors, causing increased cyclic cell responses to electrophysiological stimulation of G M P levels in principal ganglionic neurons (GREEN- the pathway from the medial septal region to hippoCARD, 1978; GREENCARD & KEBABIAN, 1974; KEBA- campal field CAI. The phosphodiesterase inhibitor isobutylmethylxanthine prolonged the time course of BIAN, 1977). Exogenous cyclic G M P and its derivatives mimic the depolarizing action of acetylcholine recovery of responses to post-tetanic stimulation. on principal ganglionic neurons of the superior cervi- Electrical stimulation of the medial septal region procal ganglion (MCAFEE& GREENGARD, 1972; DUN et duced a 2-fold increase in hippocampal cyclic G M P al., 1977a, b, 1978; GALLAGHER& SHINNICK-GAL-levels in the rabbit. It seems reasonable to infer that cyclic G M P is LAGHER,1977; HASHIGUCHI et al., 1978). ECCLES& LIBET(1961) postulated that a muscarinic cholinergic generated by activity in the cholinergic projection receptor on the principal ganglionic neuron is in- from the septum to the hippocampus in the rat. The volved in induction of the slow excitatory postsynap- cyclic G M P increases seen following stimulation pretic potential (s-EPSP) seen following preganglionic sumably occur in the pyramidal cells, although this stimulation in rabbit superior cervical ganglia. The has not been confirmed by immunohistochemistry. s-EPSP produced by preganglionic physiological Our results and those of KUHAR& YAMAMURA (1975) stimulation and blocked by atropine can be mimicked indicate that the synapses between the cholinergic by exogenous 50 PM-dibutyryl cyclic G M P (MCAFEE fibers and the pyramidal cells possess muscarinic cho& GREENGARD, 1972). LIBEThas shown that dopa- linergic receptors. Thus in the central nervous system, mine causes a marked potentiation and prolongation as in the peripheral nervous system, cyclic G M P of the s-EPSP induced in rabbit superior cervical apparently plays a role in synaptic transmission in ganglia by preganglionic stimulation. Cyclic AMP muscarinic cholinergic pathways. may have a role in potentiation of the s-EPSP (LIBET et al., 1975; KOBAYASHI et al., 1978). REFERENCES In the central nervous system, cyclic G M P causes R. E. & GREENGARD P. 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