Brain Research, 162 (1979) 231-241 (:7 Elsevier/North-Holland Biomedical Press
231
D E V E L O P M E N T OF M U S C A R I N I C C H O L I N E R G I C RECEPTORS IN INBRED STRAINS OF MICE' I D E N T I F I C A T I O N OF RECEPTOR HETEROG E N E I T Y AND RELATION TO A U D I O G E N I C SEIZURE SUSCEPTIBILITY
ROBERT S. ARONSTAM*, CAROL KELLOGG and LEO G. ABOOD Center Jbr Brain Research and Department of Psychology, University of Rochester, Rochester, N. Y. 14627 (U.S.A.)
(Accepted June 1st, 1978)
SUMMARY The concentrations and biochemical properties of muscarinic acetylcholine receptors in the brains of two highly inbred strains of mice, DBA/2J and C57BL/6J, have been studied using [3H]quinuclidinyl benzilate (QNB), a potent and specific receptor antagonist. As is the case with rat brain, murine muscarinic receptors exist in at least two forms, which differ in their affinities for receptor agonists but which have the same high affinity for receptor antagonists. Carbamylcholine binding to mouse neural membranes can be resolved into two components with Kns of 5.2 × 10-7 and 7.9 × 10-5 M. There is a regional heterogeneity of brain receptors with respect to their distribution between these high and low agonist affinity forms. Brain stem and hypothalamus receptors display binding properties that would be expected if over 60 ~ of their receptors were in the high affinity state, while only 30-40 o / o f cortex, striatum and thalamus receptors appear to be in the high affinity form. Hippocampal receptors display the least amount of high agonist affinity character. Saturation curves and Scatchard plots of QNB binding at 2, 14, 21 and 42 days postnatal age in both strains indicate no differences or changes in the affinity or nature of the binding with age. Significant increases in QNB binding per mg membrane protein were observed between 14 and 42 days in the cortex, hippocampus, striatum, thalamus and hypothalamus, but not in the midbrain-pons-medulla region. In the hippocampus the DBA mice had significantly more QNB binding. In the hypothalamus decreases with age in total binding were noted in DBA, while slight increases were noted in C57. Compared to C57, hippocampal receptors in DBA displayed lower agonist affinity at 14 and 21 days, a trait which was not apparent when DBA had * Present address: Dept. of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Md. 21201, U.S.A.
232 outgrown their audiogenic seizure sensitivity at 42 days. The differences in receptor density and agonist state distribution between the two strains may be related to audiogenie seizure sensitivity.
INTRODUCTION A number of reports have been published on the development of various components of the cholinergic neurotransmitter system in vertebrate brain, including studies of high affinity choline uptake 5,9, choline acetyltransferase 7,19 and acetylcholinesterase v. The appearance of the muscarinic receptor in brain has been studied using radiolabeled quinuclidinyl benzilate 9,H and atropinO 2 as receptor probes. In this study we consider an additional biochemical parameter of the muscarinic receptor, agonist binding characteristics. The binding of agonists, but not antagonists, deviates from a law of mass action isotherm expected for a ligand interaction with a single class of receptor z,4. To account for these anomalies, Birdsall and Hulme 4 have proposed a model of the receptor which involves the existence of two populations of receptor, which differ with respect to their affinities for receptor agonists. Antagonists have the same high affinity for the receptors in either state. We have recently demonstrated that the two receptor forms are interconvertible 2. In this study we estimate the proportion of receptors in the high and low agonist affinity states from the inhibition o f QNB binding by carbamylcholine. We demonstrate that there is a regtonal heterogeneity of neural receptors with respect to their distribution between high and low agonist forms. and that there are developmental differences in these distributions between the two inbred strains of mice studied. Inbred strains of mice showing different behavioral characteristics have been utilized to elucidate neurochemical events contributing to a specific behavior (for a review, see ref. 15). Differences in catecholamine and serotonin systems between audiogenic seizure-sensitive (such as DBA/2J) and seizure-resistant (such as C57B1 6J) strains of mice which correlate with age-dependent elaboration of seizure responses have been reported ~4. In the course of a study of the pharmacologic regulation of genetically determined seizure sensitivity, it was observed that the muscarinic agonist oxotremorine provides marked protection against seizures, even when the hypothermic effects of the drug are prevented 15. The centrally acting antagonist scopolamine prevents the protection afforded by oxotremorine, but by itself has little effect on seizure activity. Therefore, this study on the biochemical properties of the muscarinic receptor in different brain regions of seizure-sensitive and -resistant strains of mice at critical times in the development of seizure sensitivity was undertaken. METHODS Muscarinic binding was studied in mice of the C57BL/6J (audiogenic seizure-resistant, C57) and DBA/2J (audiogenic seizure-sensitive, DBA)strains at 2, 14. 21 and 42 days postnatal age. All animals were bred in the laboratory from stock originally
233 obtained from the Jackson Laboratory, Bar Harbor, Maine, and were maintained on ad libitum access to food and under diurnal lighting conditions (12-12 h light-dark cycle). The mice were killed by decapitation between 3 and 6 h after the onset of the light cycle and their brains removed and dissected into cortex, hippocampus, striatum, thalamus, hypothalamus and brain stern (midbrain, pons and medulla) fractions. Pieces were pooled from 2-6 mice in each experiment. The pieces were weighed, homogenized in 10 vols of 50 m M sodium-potassium phosphate buffer, pH 7.4, and centrifuged at 40,000 × g for 45 min. The pellets were suspended in enough buffer to yield a protein concentration of about 0.5 mg/ml. In some experiments, as noted in the text, whole brain (excluding cerebella) was used as the starting material. Protein content was determined in triplicate at two dilutions of each tissue sample by the method o f Lowry et a D v. Muscarinic binding was measured by the filtration method suggested by Yamamura and Snyder 2s. To determine the total amount of muscarinic receptor, 0.5 mg of particulate protein was incubated in a medium containing 4 n M [3H]quinuclidinyl benzilate (QNB) (16 Ci/mmole, Amersham-Searle) and 50 m M phosphate buffer, pH 7.4, in a volume of 2 ml. Under these conditions the free concentration of QNB was never reduced to below 3.3 n M by the binding. In this concentration range receptors are saturated, and specific binding is constant at all ages and in all brain areas. Specific binding was considered that binding which was blocked by the presence of 10-6 M atropine. The incubation media were incubated at 20 °C for 1 h before being filtered by suction through Whatman GF/B glass fiber filters. The filters were washed once with 7 ml ice-cold buffer, placed in plastic scintillation vials, and 10 ml of cocktail (2 liters toluene, 1 liter Triton X-100, l0 g PPO and 0.5 g POPOP) was added. After being kept in the dark for 24 h, the vials were counted in a Beckman LS 233 scintillation counter at an efficiency of 24 ~. All experimental points were determined in triplicate, including triplicate determinations of the non-specific binding. Three independent triplicate determinations of QNB binding were made on different days with mice from different litters. When QNB and carbamylcholine binding curves were performed, the concentration of receptor in the incubation media was kept below 10-1~ M by increasing the volume and decreasing the amount of membrane protein. In addition, the concentration of free QNB was adjusted by the amount of QNB that was bound. Carbamylcholine binding to the receptor was determined from its inhibition at different concentrations of the binding of 3 × 10-11 M QNB. At this concentration few receptors are occupied by QNB, and the inhibition of QNB binding becomes a good reflection of receptor occupancy by carbamylcholine 4. RESULTS In whole brain there is an increase in the amount of QNB bound per mg membrane protein with increasing age in both strains. The saturation curves are similar, and Scatchard plots of the binding data (Fig. 1) are linear and parallel, a finding which indicates no change in the affinity of the receptor for QNB with age. All
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Fig. 1. Scatchard plots of QNB binding to muscarinic receptors in neural membranes from the whole brain (except the cerebellum) of DBA (a) and C57 (b) mice at 2 ((3), 14 (Q), 21 (Q) and 4 2 ~ days postnatal age. B, pmole QNB bound per mg protein; F, concentration of unbound QNB (riM). The lines are drawn according to linear regression analyses of the data. A few points representing binding in the oldest animals are off the scale of these plots, but were included in the regression analyses. The slopes indicate dissociation constants between 2 and 4 ~ 10 10 M which do not differ significantly from each other.
dissociation constants for QNB binding are between 2 and 4 < 10-1° M [the differences are not statistically significant). Non-specific binding ranges from 5 to 22 ~ of the total binding, depending on the membrane source. The ratio o f specific to non-specific binding increases with age from an average for all brain areas of 6.7 : 1 at t4 days to 15:1 at 42 days, and is higher in forebrain areas (cortex. hippocampus, thalamus and striatum) than in brain stem areas (hypothalamus and brain stem) at all ages. There are no differences between the two strains in patterns of non-specific QNB binding. The association of QNB with muscarinic receptors is also similar in membranes from different regions of the brain. Q NB binding in different brain areas of 14-day-old DBA is indicated in Fig. 2. The differences in dissociation constants derived from these Scatchard plots are not statistically significant. Q N B binding follows the law of mass action indicating a single binding site. Specific QNB binding (4 "~ 10-9 M) in 6 brain regions as a function of age for the two strains of mice is depicted in Fig. 3. These data were subjected to analysis of variance. Sigmficant (P < 0.001) increases in binding density (binding per mg particulate protein) with age are evident in all areas except the brain stem and hypothalamus. In the hippocampus DBA have significantly (P < 0.005) greater binding, as evidenced by a significant strain difference, and in the hypothalamus a significant (P 0.03) strain by age interaction was obtained. Decreases in receptor density with age are noted in this region in DBA, while slight increases are evident in C57.
235
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Fig. 2. Scatchard plots of QNB binding to muscarinic receptors in neural membranes from brain stem ((~), hippocampus (O), cortex (D) and striatum ( m ) of immature DBA mice. B, pmole QNB bound per mg protein; F, concentration of unbound QNB (nM). The lines are drawn according to linear regression analyses• The slopes do not differ significantly.
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Fig. 3. Muscarinic receptor density (binding sites per mg membrane protein) in neural membranes from different brain regions at 14, 21 and 42 days postnatal age of DBA and C57 mice. The concentration of added QNB was 4 × 10 9 M. The points represent the average from 3 independent determinations, each performed in triplicate on different days with mice from different litters.
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Fig. 4. Inhibition of specific binding of 3 ~ tO tt M QNB by various concentrations of carbamylcholine in neural membranes from DBA and C57 mice at 3 ages. As per cent receptor occupancy by QNB decreases, the per cent blockade of QNB binding by carbamylcholine becomes a more nearly accurate reflection of receptor occupancy by carbamylcholine.
C a r b a m y l c h o l i n e i n h i b i t i o n of Q N B b i n d i n g is presented in Fig. 4. The affinity of the muscarinic receptor for c a r b a m y l c h o l i n e varies by u p t o 100-fotd i n different b r a i n regions. I n terms of receptor affinity for carbamylcholine, b r a i n stem a n d hypot h a l a m u s > ~ striatum, cortex a n d t h a l a m u s ~ hippocampus. The h i p p o c a m p u s has a
237 very low agonist affinity at 14 days in DBA and to a lesser extent in C57. Hippocampal receptor affinity for carbamylcholine increases with age in DBA so that at 42 days the affinity is the same as that of cortex, striatum and thalamus receptors. The differences between the brain stem and hypothalamus and the other brain regions are highly significant (P < 0.001) at all ages; hippocampus receptors differ significantly (P < 0.05) from cortex, thalamus and striatum receptors only at 14 days (both strains) and 21 days (DBA only). Scatcbard plots of carbamylcholine binding to mouse receptors can be resolved into two components which display dissociation constants of 5.2 ~_ 2.4 × 10-7 M and 7.9 ~ 4.2 × 10-5 M. These values are similar to those obtained with rat brain receptors". From the carbamylcholine concentration at which 50 ~ of the receptors are occupied by carbamylcholine (C50), an estimate of the fraction of receptors in the high affinity state (A) can be obtained from the sum of the mass action relationships derived for the case of two different receptors which bind the same ligand with different affinities, as follows: 1 n
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where//1 and Kz are the dissociation constants for the high and low agonist affinity receptors, respectively. The fraction of receptors displaying high affinity agonist binding in membranes from different brain areas of C57 and DBA at different ages is shown in Fig. 5. Brain stem receptors show a consistently greater high agonist affinity character in C57 than DBA, although the difference is most marked at the oldest age (P < 0.05, at 42 days). It is difficult to interpret the data from hypothalamic preparations because of the difficulty in obtaining consistent dissections of the diencephalon. The gross anatomical boundaries that separate the thalamus from the hypothalamus are not as clear as the boundaries between the other areas studied. The matter is further complicated by the great difference in agonist affinity of muscarinic receptors in the two areas. Thus, in Fig. 4, carbamylcholine binding in the hypothalamus is at times displaced from that of the brain stem, while thalamic binding is at times displaced from that of the striatum and cortex. Both displacements, however, are never seen in the same experiment. This suggests that hypothalamus and brain stem receptors, and thalamus and striatum-cortex receptors, form two groups with distinct binding properties, the deviations from this scheme occasionally observed being attributable to inaccurate subdivisions of the diencephalon. Cortical, striatal and thalamic agonist binding remain fairly constant (A 30-40 ~ ) at all ages in DBA, but display some decrease in high affinity binding with age in C57, particularly in the striatum. Hippocampal receptors in DBA increase dramatically in their high affinity agonist binding with age, a trend which is not as obvious in C57. DISCUSSION The binding of muscarinic agonists to receptors in mammalian brainl-~, 23 and
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Fig. 5. The distribution of muscarinic receptors between high and low agonist affinity states in neural membranes from various brain regions at 3 ages in DBA and C57 mice. The per cent high affinity state character was estimated from the inhibition of QNB binding by carbamylcholine as described in the text. smooth muscle s,24,26 indicates the presence of multiple binding sites. Birdsall and Hulme 4 have proposed a model to account for agonist binding properties based on the existence of two forms o f the receptor which differ with respect to their affinities for receptor agonists, antagonists binding to both forms with a uniform high affinity. Receptors can be converted from the state of low agonist affinity to one of high agonist affinity by reductive alkylation of membrane sulfhydryl groups 1,2. A simiIar pattern of agonist binding by mouse neural receptors is apparent in the results presented here. Carbamylcholine binding can be resolved into high and low affinity components with Kos of 5.2 × 10 -7 M a n d 7.9 > 10 -5 M, respectively. Agonist-antagonist competition binding curves further indicate a regional heterogeneity of brain muscarinic receptors with respect to their distribution between high and low agonist affinity states. The brain can be divided into 3 areas based on the affinities of their receptors for muscarinic agonists, namely forebrain (cerebrum, striatum, thalamus), brain stem (meduUa-pons-midbrain, hypothalamus) and hippocampal areas. Between 30 and 40 ~o of forebrain receptors are in the high agonist affinity form, while over 60 % of brain stem receptors are in the high affinity form. The fraction of receptors in the high
239 affinity state is generally lower in the hippocampus than in the other areas, although strain and age differences are evident. The apparent Ki of carbamylcholine inhibition of QNB binding ranges from 10-6 M in the brain stem to 10-4 M in 14-day DBA hippocampal receptors. The distinction between the 3 areas in agonist binding is seen with carbamylcholine, pilocarpine and arecoline in 42-day-old mice of both strains (data not presented). The amount of QNB bound per mg membrane protein increases in mouse whole brain preparations from 14 to 42 days of age. This change comes mainly from increases in the hippocampus, cortex, striatum and thalamus, where binding increases from 70 to 95 ~ of adult levels in this period. From comparisons with the data of Coyle and Yamamura 9, it is evident that the concentration of muscarinic receptors reaches adult levels at earlier ages in mouse than in rat forebrain areas. Both species show an earlier elaboration of muscarinic receptors in posterior brain regions. A number of studies have been performed on phenotypic variations of cholinergic neurocbemical parameters in DBA and C571°,18, 21. Several attempts have been made to correlate these phenotypic variations with differences in certain behavioral characteristics between the two strains. For example, compared to C57, DBA perform better on avoidance tasks and maze learning 6, display less spontaneous motor activity 20 and exhibit age-dependent sensitivity to audiogenic seizures 22. Audiogenic seizures in inbred strains of mice have provided a model in which to analyze the deve!opment of neurochemical processes in relationship to the development of a specific behaviod 5. While some DBA display seizure responses in response to sound at 14 days postnatal age, maximal response is not seen until 21 days. By 42 days, DBA show no severe seizure responses. C57 do not normally elaborate soundinduced seizures at any age. Pharmacologic studies have demonstrated that sound-induced seizures at the age of maximal sensitivity can be prevented by drugs which alter neurotransmission in several neurochemical systems 1~. In the present work, several strain differences in muscarinic acetylcholine receptors have been noted which may have an influence on seizure sensitivity. In the hippocampus, a structure which is classically related to convulsive activity, DBA have receptor densities that are significantly higher than those of C57. Moreover, a greater proportion of DBA hippocampal receptors exists in a state which has a low affinity for receptor agonists, a situation which disappears by the time DBA have outgrown their seizure susceptibility at 42 days of age. Katz and Thesleff have proposed a model of neuromuscle synapse function in which the receptor exists in high and low agonist affinity forms 1~. Based on kinetic experiments of receptor desensitization, they concluded that effective neurotransmission takes place only when an agonist binds to the low agonist affinity form of the receptor. In our studies we have found that such a model can account for several related pheomena seen with rat brain receptorsL Therefore it is not clear whether there is more or less (or the same) cholinergic influence in the hippocampus of DBA compared to C57. It is possible that the higher concentration of receptor is a compensation for the lower affinity of the existing receptors. It is also possible that the greater density and lower affinity are both reflections of more cholinergic activity.
240 ACKNOWLEDGEMENTS This work was s u p p o r t e d in part by G r a n t s DA-00464 a n d NS-10777 from the U.S. Public Health Services a n d N I M H Fellowship MH05245 to R.S.A. We wish to t h a n k Mr. D o n a l d S t e d m a n for p r e p a r a t i o n of the illustrations and Dr. M. E. Eldefrawi for his advice a n d aid in the p r e p a r a t i o n o f this manuscript.
REFERENCES 1 Aronstam, R. S., Abood, L. G. and Hoss. W., Influence of sulfhydryl reagents and heavy metals on the functional state of the muscarinic acetylcholine receptor in rat brain, Molec. Pharmacol., 14 (1978) 575-586. 2 Aronstam, R. S., Hoss, W. and Abood, L. G., Conversion between configurational states of the muscarinic receptor in rat brain, Europ. J. Pharmacol., 46 (t977) 279-282. 3 Birdsatl, N. J. M., Burgen, A. S. V., Hiley, C. R. and Wells, J. W,, Binding of agonists and antagonists to muscarinic receptors, J. supramolec. Struct., 4 (1976) 371-376. 4 Birdsall, N. J. M. and Hulme, E. C., Biochemical studies on muscarinic acetylcholine receptors, J. Neurochem., 27 (1976) 7-16. 5 Bondy, S. C. and Purdy, J. L., Development of neurotransrnitter uptake in regions of the chick brain, Brain Research, 119 (1977) 403-416. 6 Bovet, D., Bovet-Nitti, F. and Oliverio, A., Genetic aspects of learning and memory in mice, Science, 163 (1969) 139-149. 7 Burdick, C. J. and Strittmatter, C. F., Appearance of biochemical components related to acetylcholine metabolism during the embryonic development of chick brain, Arch!Biochem, 109 (1965) 293-301. 8 Burgen, A. S, V. and Hiley, C. R., Two populations of acetylcholine receptors in guinea-pig ileum, Brit. J. Pharmacol., 51 (1974) 127P. 9 Coyle, J. T. and Yamamura, H. 1., Neurochemical aspects of the ontogenesis of cholinergic neurons in the rat brain, Brain Research, 118 (1976) 429-440. 10 Ebel, A., Hermetet, J. C. and Mandel, P., Comparative study of acetylcholinesterase and choline acetyltransferase enzyme activity in brain of DBA and C57 mice, Nature New Biol., 242 (1973) 56-58. 11 Enna, S. J., Yamamura, H. 1. and Snyder, S. H., Development of muscarinic cholinergic and GABA receptor binding in chick embryo brain, Brain Research, 101 (1976) 177-183. 12 Hiley, C. R., Ontogenesis of muscarinic receptor sites in rat brain, Brit. J. Pharmacol., 53 (1976) 427P-428P. 13 Katz, B. and Thesleff, S., A study of the 'desensitization' produced by acetylcholine at the motor end-plate, J. Physiol. (Lond.), 138 (t957) 63-80. 14 Kellogg, C., Audiogenic seizures: relation to age and mechanisms of monoamineneurot ransmission, Brain Research, 106 (1976) 87-103. 15 Kellogg, C., Neurotransmitter interactions and early convulsive activity: a developmental model of behavioral responsivity. In A. Vernadakis and E. Giacobini (Eds.), Maturational Aspects of Neurotransmission Mechanisms, Karger, Basel, in press. 16 Kellogg, C. and Amaral, D., Neurotransmitter regulation of stress responses: relationship to seizure induction. In Butcher (Ed.), Cholinergic-Monoaminergic Interactions in the Brain, Academic Press, New York, in press. 17 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 18 Mandel, P., Ayad, G., Hermetet, J. C. and Ebel, A., Correlation between choline acetyltransferase activity and learning ability in different mice strains and their offspring, Brain Research; 72 (1974) 65-70. 19 Marchisio, P. C. and Giacobini, G., Choline acetyltransferase activity in the central nervous system of the developing chick, Brain Research, 15 (1969) 301-304. 20 Oliverio, A., Eleftheriou, B. E. and Bailey,D. W., Exploratory activity: genetic analysis of its modification by scopolamine and amphetamine, Physiol. Behav., 10 (1973) 893-899.
241 21 Pryor, G. T., Schlesinger, K. and Calhoun, W. H., Differences in brain enzymes among five inbred strains of mice, Life Sci., 5 (1966) 2105-2111. 22 Schlesinger, K., Boggan, W. and Freedman, D., Genetics of audiogenic seizures. 1. Relation to brain serotonin and norepinephrine in mice, Life Sci., 4 (1965) 2345 2351. 23 Snyder, S. H. and Bennett, J. P., Neurotransmitter receptors in the brain: biochemical identification, Ann. Rev. Biochem., 45 (1976) 153-175. 24 Taylor, 1. K., Cuthbert, A. W. and Young, M., Muscarinic receptors in rat intestinal muscle: comparison with the guinea pig, Europ. J. Pharmacol., 31 (1975) 319-326. 25 Yamamura, H. I. and Snyder, S. H., Muscarinic cholinergic binding in rat brain, Proc. nat. Acad. Sei. (Wash.), 71 (1974) 1725-1729. 26 Young, J. M., Desensitisation and agonist binding to cholinergic receptors in intestinal smooth muscle, FEBS Lett., 46 (1974) 354 356.
231
D E V E L O P M E N T OF M U S C A R I N I C C H O L I N E R G I C RECEPTORS IN INBRED STRAINS OF MICE' I D E N T I F I C A T I O N OF RECEPTOR HETEROG E N E I T Y AND RELATION TO A U D I O G E N I C SEIZURE SUSCEPTIBILITY
ROBERT S. ARONSTAM*, CAROL KELLOGG and LEO G. ABOOD Center Jbr Brain Research and Department of Psychology, University of Rochester, Rochester, N. Y. 14627 (U.S.A.)
(Accepted June 1st, 1978)
SUMMARY The concentrations and biochemical properties of muscarinic acetylcholine receptors in the brains of two highly inbred strains of mice, DBA/2J and C57BL/6J, have been studied using [3H]quinuclidinyl benzilate (QNB), a potent and specific receptor antagonist. As is the case with rat brain, murine muscarinic receptors exist in at least two forms, which differ in their affinities for receptor agonists but which have the same high affinity for receptor antagonists. Carbamylcholine binding to mouse neural membranes can be resolved into two components with Kns of 5.2 × 10-7 and 7.9 × 10-5 M. There is a regional heterogeneity of brain receptors with respect to their distribution between these high and low agonist affinity forms. Brain stem and hypothalamus receptors display binding properties that would be expected if over 60 ~ of their receptors were in the high affinity state, while only 30-40 o / o f cortex, striatum and thalamus receptors appear to be in the high affinity form. Hippocampal receptors display the least amount of high agonist affinity character. Saturation curves and Scatchard plots of QNB binding at 2, 14, 21 and 42 days postnatal age in both strains indicate no differences or changes in the affinity or nature of the binding with age. Significant increases in QNB binding per mg membrane protein were observed between 14 and 42 days in the cortex, hippocampus, striatum, thalamus and hypothalamus, but not in the midbrain-pons-medulla region. In the hippocampus the DBA mice had significantly more QNB binding. In the hypothalamus decreases with age in total binding were noted in DBA, while slight increases were noted in C57. Compared to C57, hippocampal receptors in DBA displayed lower agonist affinity at 14 and 21 days, a trait which was not apparent when DBA had * Present address: Dept. of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Md. 21201, U.S.A.
232 outgrown their audiogenic seizure sensitivity at 42 days. The differences in receptor density and agonist state distribution between the two strains may be related to audiogenie seizure sensitivity.
INTRODUCTION A number of reports have been published on the development of various components of the cholinergic neurotransmitter system in vertebrate brain, including studies of high affinity choline uptake 5,9, choline acetyltransferase 7,19 and acetylcholinesterase v. The appearance of the muscarinic receptor in brain has been studied using radiolabeled quinuclidinyl benzilate 9,H and atropinO 2 as receptor probes. In this study we consider an additional biochemical parameter of the muscarinic receptor, agonist binding characteristics. The binding of agonists, but not antagonists, deviates from a law of mass action isotherm expected for a ligand interaction with a single class of receptor z,4. To account for these anomalies, Birdsall and Hulme 4 have proposed a model of the receptor which involves the existence of two populations of receptor, which differ with respect to their affinities for receptor agonists. Antagonists have the same high affinity for the receptors in either state. We have recently demonstrated that the two receptor forms are interconvertible 2. In this study we estimate the proportion of receptors in the high and low agonist affinity states from the inhibition o f QNB binding by carbamylcholine. We demonstrate that there is a regtonal heterogeneity of neural receptors with respect to their distribution between high and low agonist forms. and that there are developmental differences in these distributions between the two inbred strains of mice studied. Inbred strains of mice showing different behavioral characteristics have been utilized to elucidate neurochemical events contributing to a specific behavior (for a review, see ref. 15). Differences in catecholamine and serotonin systems between audiogenic seizure-sensitive (such as DBA/2J) and seizure-resistant (such as C57B1 6J) strains of mice which correlate with age-dependent elaboration of seizure responses have been reported ~4. In the course of a study of the pharmacologic regulation of genetically determined seizure sensitivity, it was observed that the muscarinic agonist oxotremorine provides marked protection against seizures, even when the hypothermic effects of the drug are prevented 15. The centrally acting antagonist scopolamine prevents the protection afforded by oxotremorine, but by itself has little effect on seizure activity. Therefore, this study on the biochemical properties of the muscarinic receptor in different brain regions of seizure-sensitive and -resistant strains of mice at critical times in the development of seizure sensitivity was undertaken. METHODS Muscarinic binding was studied in mice of the C57BL/6J (audiogenic seizure-resistant, C57) and DBA/2J (audiogenic seizure-sensitive, DBA)strains at 2, 14. 21 and 42 days postnatal age. All animals were bred in the laboratory from stock originally
233 obtained from the Jackson Laboratory, Bar Harbor, Maine, and were maintained on ad libitum access to food and under diurnal lighting conditions (12-12 h light-dark cycle). The mice were killed by decapitation between 3 and 6 h after the onset of the light cycle and their brains removed and dissected into cortex, hippocampus, striatum, thalamus, hypothalamus and brain stern (midbrain, pons and medulla) fractions. Pieces were pooled from 2-6 mice in each experiment. The pieces were weighed, homogenized in 10 vols of 50 m M sodium-potassium phosphate buffer, pH 7.4, and centrifuged at 40,000 × g for 45 min. The pellets were suspended in enough buffer to yield a protein concentration of about 0.5 mg/ml. In some experiments, as noted in the text, whole brain (excluding cerebella) was used as the starting material. Protein content was determined in triplicate at two dilutions of each tissue sample by the method o f Lowry et a D v. Muscarinic binding was measured by the filtration method suggested by Yamamura and Snyder 2s. To determine the total amount of muscarinic receptor, 0.5 mg of particulate protein was incubated in a medium containing 4 n M [3H]quinuclidinyl benzilate (QNB) (16 Ci/mmole, Amersham-Searle) and 50 m M phosphate buffer, pH 7.4, in a volume of 2 ml. Under these conditions the free concentration of QNB was never reduced to below 3.3 n M by the binding. In this concentration range receptors are saturated, and specific binding is constant at all ages and in all brain areas. Specific binding was considered that binding which was blocked by the presence of 10-6 M atropine. The incubation media were incubated at 20 °C for 1 h before being filtered by suction through Whatman GF/B glass fiber filters. The filters were washed once with 7 ml ice-cold buffer, placed in plastic scintillation vials, and 10 ml of cocktail (2 liters toluene, 1 liter Triton X-100, l0 g PPO and 0.5 g POPOP) was added. After being kept in the dark for 24 h, the vials were counted in a Beckman LS 233 scintillation counter at an efficiency of 24 ~. All experimental points were determined in triplicate, including triplicate determinations of the non-specific binding. Three independent triplicate determinations of QNB binding were made on different days with mice from different litters. When QNB and carbamylcholine binding curves were performed, the concentration of receptor in the incubation media was kept below 10-1~ M by increasing the volume and decreasing the amount of membrane protein. In addition, the concentration of free QNB was adjusted by the amount of QNB that was bound. Carbamylcholine binding to the receptor was determined from its inhibition at different concentrations of the binding of 3 × 10-11 M QNB. At this concentration few receptors are occupied by QNB, and the inhibition of QNB binding becomes a good reflection of receptor occupancy by carbamylcholine 4. RESULTS In whole brain there is an increase in the amount of QNB bound per mg membrane protein with increasing age in both strains. The saturation curves are similar, and Scatchard plots of the binding data (Fig. 1) are linear and parallel, a finding which indicates no change in the affinity of the receptor for QNB with age. All
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Fig. 1. Scatchard plots of QNB binding to muscarinic receptors in neural membranes from the whole brain (except the cerebellum) of DBA (a) and C57 (b) mice at 2 ((3), 14 (Q), 21 (Q) and 4 2 ~ days postnatal age. B, pmole QNB bound per mg protein; F, concentration of unbound QNB (riM). The lines are drawn according to linear regression analyses of the data. A few points representing binding in the oldest animals are off the scale of these plots, but were included in the regression analyses. The slopes indicate dissociation constants between 2 and 4 ~ 10 10 M which do not differ significantly from each other.
dissociation constants for QNB binding are between 2 and 4 < 10-1° M [the differences are not statistically significant). Non-specific binding ranges from 5 to 22 ~ of the total binding, depending on the membrane source. The ratio o f specific to non-specific binding increases with age from an average for all brain areas of 6.7 : 1 at t4 days to 15:1 at 42 days, and is higher in forebrain areas (cortex. hippocampus, thalamus and striatum) than in brain stem areas (hypothalamus and brain stem) at all ages. There are no differences between the two strains in patterns of non-specific QNB binding. The association of QNB with muscarinic receptors is also similar in membranes from different regions of the brain. Q NB binding in different brain areas of 14-day-old DBA is indicated in Fig. 2. The differences in dissociation constants derived from these Scatchard plots are not statistically significant. Q N B binding follows the law of mass action indicating a single binding site. Specific QNB binding (4 "~ 10-9 M) in 6 brain regions as a function of age for the two strains of mice is depicted in Fig. 3. These data were subjected to analysis of variance. Sigmficant (P < 0.001) increases in binding density (binding per mg particulate protein) with age are evident in all areas except the brain stem and hypothalamus. In the hippocampus DBA have significantly (P < 0.005) greater binding, as evidenced by a significant strain difference, and in the hypothalamus a significant (P 0.03) strain by age interaction was obtained. Decreases in receptor density with age are noted in this region in DBA, while slight increases are evident in C57.
235
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Fig. 4. Inhibition of specific binding of 3 ~ tO tt M QNB by various concentrations of carbamylcholine in neural membranes from DBA and C57 mice at 3 ages. As per cent receptor occupancy by QNB decreases, the per cent blockade of QNB binding by carbamylcholine becomes a more nearly accurate reflection of receptor occupancy by carbamylcholine.
C a r b a m y l c h o l i n e i n h i b i t i o n of Q N B b i n d i n g is presented in Fig. 4. The affinity of the muscarinic receptor for c a r b a m y l c h o l i n e varies by u p t o 100-fotd i n different b r a i n regions. I n terms of receptor affinity for carbamylcholine, b r a i n stem a n d hypot h a l a m u s > ~ striatum, cortex a n d t h a l a m u s ~ hippocampus. The h i p p o c a m p u s has a
237 very low agonist affinity at 14 days in DBA and to a lesser extent in C57. Hippocampal receptor affinity for carbamylcholine increases with age in DBA so that at 42 days the affinity is the same as that of cortex, striatum and thalamus receptors. The differences between the brain stem and hypothalamus and the other brain regions are highly significant (P < 0.001) at all ages; hippocampus receptors differ significantly (P < 0.05) from cortex, thalamus and striatum receptors only at 14 days (both strains) and 21 days (DBA only). Scatcbard plots of carbamylcholine binding to mouse receptors can be resolved into two components which display dissociation constants of 5.2 ~_ 2.4 × 10-7 M and 7.9 ~ 4.2 × 10-5 M. These values are similar to those obtained with rat brain receptors". From the carbamylcholine concentration at which 50 ~ of the receptors are occupied by carbamylcholine (C50), an estimate of the fraction of receptors in the high affinity state (A) can be obtained from the sum of the mass action relationships derived for the case of two different receptors which bind the same ligand with different affinities, as follows: 1 n
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where//1 and Kz are the dissociation constants for the high and low agonist affinity receptors, respectively. The fraction of receptors displaying high affinity agonist binding in membranes from different brain areas of C57 and DBA at different ages is shown in Fig. 5. Brain stem receptors show a consistently greater high agonist affinity character in C57 than DBA, although the difference is most marked at the oldest age (P < 0.05, at 42 days). It is difficult to interpret the data from hypothalamic preparations because of the difficulty in obtaining consistent dissections of the diencephalon. The gross anatomical boundaries that separate the thalamus from the hypothalamus are not as clear as the boundaries between the other areas studied. The matter is further complicated by the great difference in agonist affinity of muscarinic receptors in the two areas. Thus, in Fig. 4, carbamylcholine binding in the hypothalamus is at times displaced from that of the brain stem, while thalamic binding is at times displaced from that of the striatum and cortex. Both displacements, however, are never seen in the same experiment. This suggests that hypothalamus and brain stem receptors, and thalamus and striatum-cortex receptors, form two groups with distinct binding properties, the deviations from this scheme occasionally observed being attributable to inaccurate subdivisions of the diencephalon. Cortical, striatal and thalamic agonist binding remain fairly constant (A 30-40 ~ ) at all ages in DBA, but display some decrease in high affinity binding with age in C57, particularly in the striatum. Hippocampal receptors in DBA increase dramatically in their high affinity agonist binding with age, a trend which is not as obvious in C57. DISCUSSION The binding of muscarinic agonists to receptors in mammalian brainl-~, 23 and
238
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Fig. 5. The distribution of muscarinic receptors between high and low agonist affinity states in neural membranes from various brain regions at 3 ages in DBA and C57 mice. The per cent high affinity state character was estimated from the inhibition of QNB binding by carbamylcholine as described in the text. smooth muscle s,24,26 indicates the presence of multiple binding sites. Birdsall and Hulme 4 have proposed a model to account for agonist binding properties based on the existence of two forms o f the receptor which differ with respect to their affinities for receptor agonists, antagonists binding to both forms with a uniform high affinity. Receptors can be converted from the state of low agonist affinity to one of high agonist affinity by reductive alkylation of membrane sulfhydryl groups 1,2. A simiIar pattern of agonist binding by mouse neural receptors is apparent in the results presented here. Carbamylcholine binding can be resolved into high and low affinity components with Kos of 5.2 × 10 -7 M a n d 7.9 > 10 -5 M, respectively. Agonist-antagonist competition binding curves further indicate a regional heterogeneity of brain muscarinic receptors with respect to their distribution between high and low agonist affinity states. The brain can be divided into 3 areas based on the affinities of their receptors for muscarinic agonists, namely forebrain (cerebrum, striatum, thalamus), brain stem (meduUa-pons-midbrain, hypothalamus) and hippocampal areas. Between 30 and 40 ~o of forebrain receptors are in the high agonist affinity form, while over 60 % of brain stem receptors are in the high affinity form. The fraction of receptors in the high
239 affinity state is generally lower in the hippocampus than in the other areas, although strain and age differences are evident. The apparent Ki of carbamylcholine inhibition of QNB binding ranges from 10-6 M in the brain stem to 10-4 M in 14-day DBA hippocampal receptors. The distinction between the 3 areas in agonist binding is seen with carbamylcholine, pilocarpine and arecoline in 42-day-old mice of both strains (data not presented). The amount of QNB bound per mg membrane protein increases in mouse whole brain preparations from 14 to 42 days of age. This change comes mainly from increases in the hippocampus, cortex, striatum and thalamus, where binding increases from 70 to 95 ~ of adult levels in this period. From comparisons with the data of Coyle and Yamamura 9, it is evident that the concentration of muscarinic receptors reaches adult levels at earlier ages in mouse than in rat forebrain areas. Both species show an earlier elaboration of muscarinic receptors in posterior brain regions. A number of studies have been performed on phenotypic variations of cholinergic neurocbemical parameters in DBA and C571°,18, 21. Several attempts have been made to correlate these phenotypic variations with differences in certain behavioral characteristics between the two strains. For example, compared to C57, DBA perform better on avoidance tasks and maze learning 6, display less spontaneous motor activity 20 and exhibit age-dependent sensitivity to audiogenic seizures 22. Audiogenic seizures in inbred strains of mice have provided a model in which to analyze the deve!opment of neurochemical processes in relationship to the development of a specific behaviod 5. While some DBA display seizure responses in response to sound at 14 days postnatal age, maximal response is not seen until 21 days. By 42 days, DBA show no severe seizure responses. C57 do not normally elaborate soundinduced seizures at any age. Pharmacologic studies have demonstrated that sound-induced seizures at the age of maximal sensitivity can be prevented by drugs which alter neurotransmission in several neurochemical systems 1~. In the present work, several strain differences in muscarinic acetylcholine receptors have been noted which may have an influence on seizure sensitivity. In the hippocampus, a structure which is classically related to convulsive activity, DBA have receptor densities that are significantly higher than those of C57. Moreover, a greater proportion of DBA hippocampal receptors exists in a state which has a low affinity for receptor agonists, a situation which disappears by the time DBA have outgrown their seizure susceptibility at 42 days of age. Katz and Thesleff have proposed a model of neuromuscle synapse function in which the receptor exists in high and low agonist affinity forms 1~. Based on kinetic experiments of receptor desensitization, they concluded that effective neurotransmission takes place only when an agonist binds to the low agonist affinity form of the receptor. In our studies we have found that such a model can account for several related pheomena seen with rat brain receptorsL Therefore it is not clear whether there is more or less (or the same) cholinergic influence in the hippocampus of DBA compared to C57. It is possible that the higher concentration of receptor is a compensation for the lower affinity of the existing receptors. It is also possible that the greater density and lower affinity are both reflections of more cholinergic activity.
240 ACKNOWLEDGEMENTS This work was s u p p o r t e d in part by G r a n t s DA-00464 a n d NS-10777 from the U.S. Public Health Services a n d N I M H Fellowship MH05245 to R.S.A. We wish to t h a n k Mr. D o n a l d S t e d m a n for p r e p a r a t i o n of the illustrations and Dr. M. E. Eldefrawi for his advice a n d aid in the p r e p a r a t i o n o f this manuscript.
REFERENCES 1 Aronstam, R. S., Abood, L. G. and Hoss. W., Influence of sulfhydryl reagents and heavy metals on the functional state of the muscarinic acetylcholine receptor in rat brain, Molec. Pharmacol., 14 (1978) 575-586. 2 Aronstam, R. S., Hoss, W. and Abood, L. G., Conversion between configurational states of the muscarinic receptor in rat brain, Europ. J. Pharmacol., 46 (t977) 279-282. 3 Birdsatl, N. J. M., Burgen, A. S. V., Hiley, C. R. and Wells, J. W,, Binding of agonists and antagonists to muscarinic receptors, J. supramolec. Struct., 4 (1976) 371-376. 4 Birdsall, N. J. M. and Hulme, E. C., Biochemical studies on muscarinic acetylcholine receptors, J. Neurochem., 27 (1976) 7-16. 5 Bondy, S. C. and Purdy, J. L., Development of neurotransrnitter uptake in regions of the chick brain, Brain Research, 119 (1977) 403-416. 6 Bovet, D., Bovet-Nitti, F. and Oliverio, A., Genetic aspects of learning and memory in mice, Science, 163 (1969) 139-149. 7 Burdick, C. J. and Strittmatter, C. F., Appearance of biochemical components related to acetylcholine metabolism during the embryonic development of chick brain, Arch!Biochem, 109 (1965) 293-301. 8 Burgen, A. S, V. and Hiley, C. R., Two populations of acetylcholine receptors in guinea-pig ileum, Brit. J. Pharmacol., 51 (1974) 127P. 9 Coyle, J. T. and Yamamura, H. 1., Neurochemical aspects of the ontogenesis of cholinergic neurons in the rat brain, Brain Research, 118 (1976) 429-440. 10 Ebel, A., Hermetet, J. C. and Mandel, P., Comparative study of acetylcholinesterase and choline acetyltransferase enzyme activity in brain of DBA and C57 mice, Nature New Biol., 242 (1973) 56-58. 11 Enna, S. J., Yamamura, H. 1. and Snyder, S. H., Development of muscarinic cholinergic and GABA receptor binding in chick embryo brain, Brain Research, 101 (1976) 177-183. 12 Hiley, C. R., Ontogenesis of muscarinic receptor sites in rat brain, Brit. J. Pharmacol., 53 (1976) 427P-428P. 13 Katz, B. and Thesleff, S., A study of the 'desensitization' produced by acetylcholine at the motor end-plate, J. Physiol. (Lond.), 138 (t957) 63-80. 14 Kellogg, C., Audiogenic seizures: relation to age and mechanisms of monoamineneurot ransmission, Brain Research, 106 (1976) 87-103. 15 Kellogg, C., Neurotransmitter interactions and early convulsive activity: a developmental model of behavioral responsivity. In A. Vernadakis and E. Giacobini (Eds.), Maturational Aspects of Neurotransmission Mechanisms, Karger, Basel, in press. 16 Kellogg, C. and Amaral, D., Neurotransmitter regulation of stress responses: relationship to seizure induction. In Butcher (Ed.), Cholinergic-Monoaminergic Interactions in the Brain, Academic Press, New York, in press. 17 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 18 Mandel, P., Ayad, G., Hermetet, J. C. and Ebel, A., Correlation between choline acetyltransferase activity and learning ability in different mice strains and their offspring, Brain Research; 72 (1974) 65-70. 19 Marchisio, P. C. and Giacobini, G., Choline acetyltransferase activity in the central nervous system of the developing chick, Brain Research, 15 (1969) 301-304. 20 Oliverio, A., Eleftheriou, B. E. and Bailey,D. W., Exploratory activity: genetic analysis of its modification by scopolamine and amphetamine, Physiol. Behav., 10 (1973) 893-899.
241 21 Pryor, G. T., Schlesinger, K. and Calhoun, W. H., Differences in brain enzymes among five inbred strains of mice, Life Sci., 5 (1966) 2105-2111. 22 Schlesinger, K., Boggan, W. and Freedman, D., Genetics of audiogenic seizures. 1. Relation to brain serotonin and norepinephrine in mice, Life Sci., 4 (1965) 2345 2351. 23 Snyder, S. H. and Bennett, J. P., Neurotransmitter receptors in the brain: biochemical identification, Ann. Rev. Biochem., 45 (1976) 153-175. 24 Taylor, 1. K., Cuthbert, A. W. and Young, M., Muscarinic receptors in rat intestinal muscle: comparison with the guinea pig, Europ. J. Pharmacol., 31 (1975) 319-326. 25 Yamamura, H. I. and Snyder, S. H., Muscarinic cholinergic binding in rat brain, Proc. nat. Acad. Sei. (Wash.), 71 (1974) 1725-1729. 26 Young, J. M., Desensitisation and agonist binding to cholinergic receptors in intestinal smooth muscle, FEBS Lett., 46 (1974) 354 356.
