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8-Br-cyclic GMP mimics activation of muscarinic autoreceptor and inhibits acetylcholine release from rat hippocampal slices
Brain Research, 213 (1981) 467-471 © Elsevier/North-Holland Biomedical Press
467
8-Br-cyclic GMP mimics activation of muscarinic autoreceptor and inhibits acetylcholine release from rat hippocampal slices
OIE NORDSTROM and TAMAS BARTFAI Department of Biochemistry, ,4rrhenius Laboratory, University of Stockholm, S-106 91 Stockholm ( S wedenJ
(Accepted January 15th, 1981) Key words: muscarinic receptor -- autoreceptor - - acetylcholine release - - cyclic GMP - - hippo-
campus -- acetylcholine
In the rat hippocampus acetylcholine depresses its own release via stimulation of muscarinic acetylcholine receptors12,13. Experiments carried out, in the presence of tetrodotoxin, on a suspension of isolated nerve-endings from hippocampus indicated that the muscarinic feedback regulation is exerted without activation of neuronal loops 1°. It was concluded that muscarinic autoreceptors localized on cholinergic nerveendings may mediate inhibition of the release of [aH]acetylcholine ([3H]ACh)I°. Stimulation of muscarinic receptors on the periphery and in the central nervous system leads to increases in cyclic G M P levels2, 6,9. The present study addresses the question of whether the effects of cholinergic agonists, at the muscarinic autoreceptor, are mediated by cyclic GMP. Preliminary experiments with dibutyryl cyclic G M P indicated that this may be the case 1. Rat hippocampi were cut into 0,4 mm thick slices by a Mcllwain tissue chopper in sagittal direction. The slices were incubated with 1/~M choline and with [3H]choline ([3H]Ch) 50 nM as a tracer 1°, in Krebs-Ringer's buffer (NaC1, 138 mM; KC1, 5 raM; MgC12, 1 raM; NaH~PO4, 1 mM; NaHCO3, 11 mM; glucose, 10 mM NaOOCCHa, 1 mM; and CaC12, 2 mM) for 40 mio at 37 °C under continuous bubbling with 95 ~ 02 and 5 ~ COz. When Krebs-Ringer's buffer with 25 mM K ÷ was used (instead of 5 mM K ÷) the Na + concentration was lowered by 20 mM to maintain iso-osmolarity. The evoked (by K ÷ 25 mM) release of [aH]ACh and [aH]Ch and the effect of drugs on it was studied for 5 min at 37 °C. [3H]ACh and [3H]Ch were separated by heptanon extraction s of [aH]ACh. The latter was counted after removal of the solvent in a Beckman scintillation spectrometer in Lumagel, from Lumac System AG, Basel. Cyclic G M P was extracted from the slices used in the experiments on [3H]ACh release according to Folbergrov~iS. One ml perchloric acid (0.3 M) containing EDTA (1 raM) was added to each slice ( < 10 mg protein) and the samples were rapidly frozen on dryice. After thawing the samples, 0.1 ml HCI (0.1 M), in methanol was added and the samples were sonicated. The sonicate was centrifuged and the protein pellet, preeipita-
468 ted by perchloric acid, was used for protein determination according to Lowry et al. 7. The perchlorate ions in the supernatant were precipitated by addition of 85/tl K O H (5 M). The resulting KC104 pellet was discarded, and the p H of the supernatant was adjusted to p H 6 before withdrawing aliquots for an acetylated cyclic G M P radioimmunoassay 4. Tritium labeled choline (methyl-[aH]choline) chloride, 84 Ci/mmol, was purchased from the Radiochemical Center, Amersham. 3-Isobutyl-l-methylxanthine (IBMX), 8-Br-cyclic AMP, 8-Br-cyclic GMP, were bought from Sigma Chemicals, St. Louis, Miss. Oxotremorine was a product of E G A AG, Steinheim. Atropine was bought from Merck, Darmstad. Other chemicals were of analytical grade. Raising K + concentration from 5 m M to 25 mM in the presence of the phosphodiesterase inhibitor, IBMX (1 mM) led to an increase in the cyclic G M P content of the hippocampal slices from 0.26 to 0.95 pmol/mg protein. This finding is in agreement with previous findings on K+-depolarization mediated increases in cyclic G M P levels in nervous tissue2,6,11. Atropine reduced by 60 % the K ÷ (25 mM) evoked increase in cyclic G M P levels (Fig. 1) indicating that this portion of the increase may reflect stimulation of muscarinic receptors, which are known to activate cyclic G M P synthesis 2,a,11. In the hippocampus, oxotremorine a muscarinic agonist can raise cyclic G M P levels 3, also in the absence of K+-depolarization. Under our experimental conditions (K ÷ 5 mM) this
1.0-
c a~ o ca. ol
-x:
E
~_ 0.5u
o E
K÷: 5 m M
K~25mM Atr
Ox0
-. At r + oxo
Fig. 1. Increases in cyclic GMP levels in rat hippocampal slices incubated for 5 min at 37 ~C with Krebs-Ringer's buffer containing K + (25 raM) in the absence and presence of atropine 1/~M (Atr), oxotremorine 10 /~M (Oxo) or both. In all experiments 3-isobutyl-l-methylxanthine (1 mM) and eserine (10/zM) were present. ** P < 0.05 significantly different from control (K+:25 mM). All data are given as mean ± S.D. for 3-6 samples each assayed in triplicates. "Basal" cyclic GMP level found in K + (5 mM) after incubation with 3-isobutyl-l-methylxanthine was 0.26 ± 0.04 pmol/mg protein.
469 increase is from 0.26 to 0.6 pmol/mg protein. Oxotremorine when added in the presence of high K + (25 mM) concentration, could antagonize the effects of atropine (,Fig. 1), but could not further activate cyclic G M P synthesis activated by K + depolarization. This result may be interpreted as indicative of a close to full activation of muscarinic receptors by the endogenous ACh which is released by K + (25 mM) depolarization. These experiments indicated that cyclic G M P synthesis in hippocampal slices could be stimulated via muscarinic receptors. This made it plausible to ask the question whether cyclic G M P participates in mediating the action of ACh at the presynaptic muscarinic receptors. Unfortunately we could not evaluate how large a portion of the elevated cyclic G M P levels originated from activation of presynaptic and postsynaptic muscarinic receptors, respectively. Lesion studies with kainic acid (unpublished results) or septal lesion 14 failed to give answer to this question. The latter was shown 14 to change the number of muscarinic receptors in hippocampus only negligibly. Therefore, penetrating analogues of cyclic GMP, 8-Br-cyclic G M P and dibutyryl cyclic G M P were used to examine whether cyclic G M P is involved in regulation ofacetylcholine release. 8-Br-cyclic G M P (200 #M) and dibutyryl-cyclic G M P (200 y M ) but not 8-Brcyclic AMP (200 y M ) inhibited release of [3H]ACh from slices loaded with [sH]Ch (Fig. 2). This nucleotide specificity is in line with previous findings on muscarinic receptor-cyclic G M P coupling in nervous tissue and in smooth muscle2, 3,9,11. Atro-
10-
c E u~
;
c
o
[×x
E
Ixx
l
lx~ lxx
m E o_ r(..)
ff
i
r
K ÷: 5 mM Oxo
8-BrcGMP
Oxo* 8-Br-cGMP
K+: 25 mM Atr.* Atr. At~ + 8-Br-cGMP oxo
db-cGMP 8-Br-cAMP
Fig. 2. The effect of muscarinic drugs and cyclic nucleotide analogues on evoked release (K+ 25 mM) of [3H]acetylcholine(pmol/mg protein/5 rain) from hippocampal slices. All values are presented as mean ± S.D. for 3-6 experiments. The abbreviations Oxo, 8-Br-cyclic GMP; dbcGMP, Atr, 8-BrcAMP stand for oxotremorine (10 #M), 8-Br-cyclicGMP (200/~M) dibutyryl cyclic GMP (200/~M), atropine (1 yM) and 8-Br-cyclic AMP (200 /~M), respectively. The drugs were present for 5 min together with the high K + (25 mM) containing Krebs-Ringer's buffer. Eserine (10/~M) was present throughout the whole experiment.
470
E 6 Ln
f
~
K~25 rnM
E a_ 2
~K~SmM ) ~-o
2;o
3;0
~o
sb0
Fig. 3. Dose dependenceof the inhibitoryeffectof 8-Br-cyclicGMP on release of pH]acetylcholine from hippocampal slices. Conditionsas in Fig. 2. pine (1 #M) could abolish inhibition of pH]ACh release by endogenous acetylcholine (accumulated in the presence of eserine 10 #M). Atropine (1/,M) was, however, without effect when 8-Br-cyclic GMP (200 #M) was used to inhibit the release of pH]ACh. This result indicated that the cyclic GMP analogue bypassed the muscarinic autoreceptor. 8-Br-cyclic GMP was more effective in inhibiting evoked release (25 mM K ÷) of pH]ACh than in inhibiting basal release or leakage (5 mM K ÷) (Fig. 3). The effects of 8-Br-cyclic GMP were dose-dependent and reached maximum at a concentration of 200 HM (Fig. 3). 8-Br-Cyclic GMP (200 /~M) which has maximal effect inhibits release of pH]ACh to the same extent as saturating oxotremorine (10 #M); furthermore it was noted that 8-Br-cyclic GMP (200/,M) did not potentiate the inhibition of release of pH]ACh by oxotremorine (10 #M) (Fig. 2). One explanation for this finding might be that the inhibition caused by oxotremorine or 8-Br-cyclic GMP alone was maximal already and that the two compounds act by a common mechanism. In fact previous studies with muscarinic agonists10, indicated that the maximal inhibition of pH]ACh release by the autoreceptor is about 30 % of the evoked release 3. (Efflux of pH]Ch evoked by high K ÷ (25 mM) was higher by 30 % than in normal Krebs-Ringer's buffer (K+: 5 mM) but was virtually unaffected by muscarinic drugs 10 and cyclic nucleotide derivatives.) The above results indicate that muscarinic receptors regulate cyclic GMP synthesis in hippocampal slices. The experiments with penetrating cyclic GMP analogues indicate also that cyclic GMP might act as a second messenger when the muscarinic autoreceptor is activated. Similar conclusions were reached by experiments on cerebral cortex during the time this manuscript was prepared 15. This work was supported from grants by National Institute of Mental Health, Bethesda and the Swedish Medical Research Council.
471 1 Bartfai, T., Nordstr6m, O. and Tj6rnhammar, M. L., Cyclic guanosine 3'5'-monophosphate in the nervous system: pre-, post- and transsynaptic effects, Progr. PharmacoL, 4 (1980) 151-157. 2 Bartfai, T., Study, R. E. and Greengard, P., Muscarinic stimulation and cGMP synthesis in the nervous system. In D. J. Jenden (Ed.), Cholinergic Mechanisms and Psychopharmacology, Plenum Press, New York, 1977, pp. 285-295. 3 Black, Jr., A. C., Sandquist, D., West, J. R., Wamsley, J. K. and Williams, T. H., Muscarinic cholinergic stimulation increases cyclic GMP levels in rat hippocampus, J. Neurochem., 33 (1979) 1165-1168. 4 Brooker, G., Harper, J. F., Teraski, W. L. and Moylan, R. D., Radioimmunoassay of cyclic AMP and cyclic GMP. In G. Brooker, P. Greengard and G. A. Robinson (Eds.), Advances in Cyclic Nucleotide Research, Vol. 10, Raven Press, New York, 1979, pp. 1-33. 5 Folbergrov~i, J., Cyclic 3'5"-adenosine monophosphate in mouse cerebral cortex during homocysteine convulsions and their prevention by sodium phenobarbital, Brain Research, 92 (1975) 165-169.
6 Kinscherf, D. A., Chang, M. M., Rubin, E. M., Schneider, D. R. and Ferrendelli, J. A., Comparison of the effects of depolarizing agents and neurotransmitterson regional CNS cyclic GMP levels in various animals, J. Neurochem., 26 (1976) 527-530. 7 Lowry, O. I-I., Rosebrough, V. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-270. 8 McGee, R., Simpson, P., Christian, C. Mata, M., Nelson, P. and Nirenberg, M., Regulation of acetylcholine release from neuroblastoma x glioma hybrid cells, Proc. nat. Acad. Sci. (Wash.), 75 (1978) 1314-1318. 9 Nathanson, J. A., Cyclic nucleotides and central nervous system function, Physiol. Rev., 57 (1977) 157-256. 10 Nordstr6m, O. and Bartfai, T., Muscarinic autoreceptor regulates acetylcholine release in rat hippocampus: in vitro evidence, Acta physiol, scand., 108 (1980) 347-353. 11 Study, R. E., Breakefield, X. O., Bartfai, T. and Greengard, P., Voltage-sensitive calcium channels regulate guanosine 3'5'-cyclic monophosphate levels in neuroblastoma cells, Proc. nat. Acad. Sci. (Wash.), 75 (1978) 6295-6299. 12 Szerb, J. C., Characterization of presynaptic muscarinic receptors in central cholinergic neurons. In D. J. Jenden (Ed.), Cholinergic Mechansims andPsychopharmacology, Plenum Press, New York, 1977, pp. 49-60. 13 Szerb, J., C., Hadhhzy, P. and Dudar, J. D., Release of [ZH]acetylcholinefrom rat hippocampal slices: effect of septal lesion and of graded concentration of muscarinic agonists and antagonists, Brain Research, 128 (1977) 285-291. 14 Yamamura, H. J. and Snyder, S. H., Postsynaptic localization of muscarinic cholinergic receptor binding in rat hippocampus, Brain Research, 78 (1974) 320-326. 15 Yonehara, N., Matsuda, T., Saito, K., lshida, H. and Yoshida, H., Effect of cyclic nucleotide derivatives on the release of ACh from cortical slices of the rat brain, Brain Research, 182 (1980) 137-144.
467
8-Br-cyclic GMP mimics activation of muscarinic autoreceptor and inhibits acetylcholine release from rat hippocampal slices
OIE NORDSTROM and TAMAS BARTFAI Department of Biochemistry, ,4rrhenius Laboratory, University of Stockholm, S-106 91 Stockholm ( S wedenJ
(Accepted January 15th, 1981) Key words: muscarinic receptor -- autoreceptor - - acetylcholine release - - cyclic GMP - - hippo-
campus -- acetylcholine
In the rat hippocampus acetylcholine depresses its own release via stimulation of muscarinic acetylcholine receptors12,13. Experiments carried out, in the presence of tetrodotoxin, on a suspension of isolated nerve-endings from hippocampus indicated that the muscarinic feedback regulation is exerted without activation of neuronal loops 1°. It was concluded that muscarinic autoreceptors localized on cholinergic nerveendings may mediate inhibition of the release of [aH]acetylcholine ([3H]ACh)I°. Stimulation of muscarinic receptors on the periphery and in the central nervous system leads to increases in cyclic G M P levels2, 6,9. The present study addresses the question of whether the effects of cholinergic agonists, at the muscarinic autoreceptor, are mediated by cyclic GMP. Preliminary experiments with dibutyryl cyclic G M P indicated that this may be the case 1. Rat hippocampi were cut into 0,4 mm thick slices by a Mcllwain tissue chopper in sagittal direction. The slices were incubated with 1/~M choline and with [3H]choline ([3H]Ch) 50 nM as a tracer 1°, in Krebs-Ringer's buffer (NaC1, 138 mM; KC1, 5 raM; MgC12, 1 raM; NaH~PO4, 1 mM; NaHCO3, 11 mM; glucose, 10 mM NaOOCCHa, 1 mM; and CaC12, 2 mM) for 40 mio at 37 °C under continuous bubbling with 95 ~ 02 and 5 ~ COz. When Krebs-Ringer's buffer with 25 mM K ÷ was used (instead of 5 mM K ÷) the Na + concentration was lowered by 20 mM to maintain iso-osmolarity. The evoked (by K ÷ 25 mM) release of [aH]ACh and [aH]Ch and the effect of drugs on it was studied for 5 min at 37 °C. [3H]ACh and [3H]Ch were separated by heptanon extraction s of [aH]ACh. The latter was counted after removal of the solvent in a Beckman scintillation spectrometer in Lumagel, from Lumac System AG, Basel. Cyclic G M P was extracted from the slices used in the experiments on [3H]ACh release according to Folbergrov~iS. One ml perchloric acid (0.3 M) containing EDTA (1 raM) was added to each slice ( < 10 mg protein) and the samples were rapidly frozen on dryice. After thawing the samples, 0.1 ml HCI (0.1 M), in methanol was added and the samples were sonicated. The sonicate was centrifuged and the protein pellet, preeipita-
468 ted by perchloric acid, was used for protein determination according to Lowry et al. 7. The perchlorate ions in the supernatant were precipitated by addition of 85/tl K O H (5 M). The resulting KC104 pellet was discarded, and the p H of the supernatant was adjusted to p H 6 before withdrawing aliquots for an acetylated cyclic G M P radioimmunoassay 4. Tritium labeled choline (methyl-[aH]choline) chloride, 84 Ci/mmol, was purchased from the Radiochemical Center, Amersham. 3-Isobutyl-l-methylxanthine (IBMX), 8-Br-cyclic AMP, 8-Br-cyclic GMP, were bought from Sigma Chemicals, St. Louis, Miss. Oxotremorine was a product of E G A AG, Steinheim. Atropine was bought from Merck, Darmstad. Other chemicals were of analytical grade. Raising K + concentration from 5 m M to 25 mM in the presence of the phosphodiesterase inhibitor, IBMX (1 mM) led to an increase in the cyclic G M P content of the hippocampal slices from 0.26 to 0.95 pmol/mg protein. This finding is in agreement with previous findings on K+-depolarization mediated increases in cyclic G M P levels in nervous tissue2,6,11. Atropine reduced by 60 % the K ÷ (25 mM) evoked increase in cyclic G M P levels (Fig. 1) indicating that this portion of the increase may reflect stimulation of muscarinic receptors, which are known to activate cyclic G M P synthesis 2,a,11. In the hippocampus, oxotremorine a muscarinic agonist can raise cyclic G M P levels 3, also in the absence of K+-depolarization. Under our experimental conditions (K ÷ 5 mM) this
1.0-
c a~ o ca. ol
-x:
E
~_ 0.5u
o E
K÷: 5 m M
K~25mM Atr
Ox0
-. At r + oxo
Fig. 1. Increases in cyclic GMP levels in rat hippocampal slices incubated for 5 min at 37 ~C with Krebs-Ringer's buffer containing K + (25 raM) in the absence and presence of atropine 1/~M (Atr), oxotremorine 10 /~M (Oxo) or both. In all experiments 3-isobutyl-l-methylxanthine (1 mM) and eserine (10/zM) were present. ** P < 0.05 significantly different from control (K+:25 mM). All data are given as mean ± S.D. for 3-6 samples each assayed in triplicates. "Basal" cyclic GMP level found in K + (5 mM) after incubation with 3-isobutyl-l-methylxanthine was 0.26 ± 0.04 pmol/mg protein.
469 increase is from 0.26 to 0.6 pmol/mg protein. Oxotremorine when added in the presence of high K + (25 mM) concentration, could antagonize the effects of atropine (,Fig. 1), but could not further activate cyclic G M P synthesis activated by K + depolarization. This result may be interpreted as indicative of a close to full activation of muscarinic receptors by the endogenous ACh which is released by K + (25 mM) depolarization. These experiments indicated that cyclic G M P synthesis in hippocampal slices could be stimulated via muscarinic receptors. This made it plausible to ask the question whether cyclic G M P participates in mediating the action of ACh at the presynaptic muscarinic receptors. Unfortunately we could not evaluate how large a portion of the elevated cyclic G M P levels originated from activation of presynaptic and postsynaptic muscarinic receptors, respectively. Lesion studies with kainic acid (unpublished results) or septal lesion 14 failed to give answer to this question. The latter was shown 14 to change the number of muscarinic receptors in hippocampus only negligibly. Therefore, penetrating analogues of cyclic GMP, 8-Br-cyclic G M P and dibutyryl cyclic G M P were used to examine whether cyclic G M P is involved in regulation ofacetylcholine release. 8-Br-cyclic G M P (200 #M) and dibutyryl-cyclic G M P (200 y M ) but not 8-Brcyclic AMP (200 y M ) inhibited release of [3H]ACh from slices loaded with [sH]Ch (Fig. 2). This nucleotide specificity is in line with previous findings on muscarinic receptor-cyclic G M P coupling in nervous tissue and in smooth muscle2, 3,9,11. Atro-
10-
c E u~
;
c
o
[×x
E
Ixx
l
lx~ lxx
m E o_ r(..)
ff
i
r
K ÷: 5 mM Oxo
8-BrcGMP
Oxo* 8-Br-cGMP
K+: 25 mM Atr.* Atr. At~ + 8-Br-cGMP oxo
db-cGMP 8-Br-cAMP
Fig. 2. The effect of muscarinic drugs and cyclic nucleotide analogues on evoked release (K+ 25 mM) of [3H]acetylcholine(pmol/mg protein/5 rain) from hippocampal slices. All values are presented as mean ± S.D. for 3-6 experiments. The abbreviations Oxo, 8-Br-cyclic GMP; dbcGMP, Atr, 8-BrcAMP stand for oxotremorine (10 #M), 8-Br-cyclicGMP (200/~M) dibutyryl cyclic GMP (200/~M), atropine (1 yM) and 8-Br-cyclic AMP (200 /~M), respectively. The drugs were present for 5 min together with the high K + (25 mM) containing Krebs-Ringer's buffer. Eserine (10/~M) was present throughout the whole experiment.
470
E 6 Ln
f
~
K~25 rnM
E a_ 2
~K~SmM ) ~-o
2;o
3;0
~o
sb0
Fig. 3. Dose dependenceof the inhibitoryeffectof 8-Br-cyclicGMP on release of pH]acetylcholine from hippocampal slices. Conditionsas in Fig. 2. pine (1 #M) could abolish inhibition of pH]ACh release by endogenous acetylcholine (accumulated in the presence of eserine 10 #M). Atropine (1/,M) was, however, without effect when 8-Br-cyclic GMP (200 #M) was used to inhibit the release of pH]ACh. This result indicated that the cyclic GMP analogue bypassed the muscarinic autoreceptor. 8-Br-cyclic GMP was more effective in inhibiting evoked release (25 mM K ÷) of pH]ACh than in inhibiting basal release or leakage (5 mM K ÷) (Fig. 3). The effects of 8-Br-cyclic GMP were dose-dependent and reached maximum at a concentration of 200 HM (Fig. 3). 8-Br-Cyclic GMP (200 /~M) which has maximal effect inhibits release of pH]ACh to the same extent as saturating oxotremorine (10 #M); furthermore it was noted that 8-Br-cyclic GMP (200/,M) did not potentiate the inhibition of release of pH]ACh by oxotremorine (10 #M) (Fig. 2). One explanation for this finding might be that the inhibition caused by oxotremorine or 8-Br-cyclic GMP alone was maximal already and that the two compounds act by a common mechanism. In fact previous studies with muscarinic agonists10, indicated that the maximal inhibition of pH]ACh release by the autoreceptor is about 30 % of the evoked release 3. (Efflux of pH]Ch evoked by high K ÷ (25 mM) was higher by 30 % than in normal Krebs-Ringer's buffer (K+: 5 mM) but was virtually unaffected by muscarinic drugs 10 and cyclic nucleotide derivatives.) The above results indicate that muscarinic receptors regulate cyclic GMP synthesis in hippocampal slices. The experiments with penetrating cyclic GMP analogues indicate also that cyclic GMP might act as a second messenger when the muscarinic autoreceptor is activated. Similar conclusions were reached by experiments on cerebral cortex during the time this manuscript was prepared 15. This work was supported from grants by National Institute of Mental Health, Bethesda and the Swedish Medical Research Council.
471 1 Bartfai, T., Nordstr6m, O. and Tj6rnhammar, M. L., Cyclic guanosine 3'5'-monophosphate in the nervous system: pre-, post- and transsynaptic effects, Progr. PharmacoL, 4 (1980) 151-157. 2 Bartfai, T., Study, R. E. and Greengard, P., Muscarinic stimulation and cGMP synthesis in the nervous system. In D. J. Jenden (Ed.), Cholinergic Mechanisms and Psychopharmacology, Plenum Press, New York, 1977, pp. 285-295. 3 Black, Jr., A. C., Sandquist, D., West, J. R., Wamsley, J. K. and Williams, T. H., Muscarinic cholinergic stimulation increases cyclic GMP levels in rat hippocampus, J. Neurochem., 33 (1979) 1165-1168. 4 Brooker, G., Harper, J. F., Teraski, W. L. and Moylan, R. D., Radioimmunoassay of cyclic AMP and cyclic GMP. In G. Brooker, P. Greengard and G. A. Robinson (Eds.), Advances in Cyclic Nucleotide Research, Vol. 10, Raven Press, New York, 1979, pp. 1-33. 5 Folbergrov~i, J., Cyclic 3'5"-adenosine monophosphate in mouse cerebral cortex during homocysteine convulsions and their prevention by sodium phenobarbital, Brain Research, 92 (1975) 165-169.
6 Kinscherf, D. A., Chang, M. M., Rubin, E. M., Schneider, D. R. and Ferrendelli, J. A., Comparison of the effects of depolarizing agents and neurotransmitterson regional CNS cyclic GMP levels in various animals, J. Neurochem., 26 (1976) 527-530. 7 Lowry, O. I-I., Rosebrough, V. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-270. 8 McGee, R., Simpson, P., Christian, C. Mata, M., Nelson, P. and Nirenberg, M., Regulation of acetylcholine release from neuroblastoma x glioma hybrid cells, Proc. nat. Acad. Sci. (Wash.), 75 (1978) 1314-1318. 9 Nathanson, J. A., Cyclic nucleotides and central nervous system function, Physiol. Rev., 57 (1977) 157-256. 10 Nordstr6m, O. and Bartfai, T., Muscarinic autoreceptor regulates acetylcholine release in rat hippocampus: in vitro evidence, Acta physiol, scand., 108 (1980) 347-353. 11 Study, R. E., Breakefield, X. O., Bartfai, T. and Greengard, P., Voltage-sensitive calcium channels regulate guanosine 3'5'-cyclic monophosphate levels in neuroblastoma cells, Proc. nat. Acad. Sci. (Wash.), 75 (1978) 6295-6299. 12 Szerb, J. C., Characterization of presynaptic muscarinic receptors in central cholinergic neurons. In D. J. Jenden (Ed.), Cholinergic Mechansims andPsychopharmacology, Plenum Press, New York, 1977, pp. 49-60. 13 Szerb, J., C., Hadhhzy, P. and Dudar, J. D., Release of [ZH]acetylcholinefrom rat hippocampal slices: effect of septal lesion and of graded concentration of muscarinic agonists and antagonists, Brain Research, 128 (1977) 285-291. 14 Yamamura, H. J. and Snyder, S. H., Postsynaptic localization of muscarinic cholinergic receptor binding in rat hippocampus, Brain Research, 78 (1974) 320-326. 15 Yonehara, N., Matsuda, T., Saito, K., lshida, H. and Yoshida, H., Effect of cyclic nucleotide derivatives on the release of ACh from cortical slices of the rat brain, Brain Research, 182 (1980) 137-144.