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Nicotinic cholinergic receptors in rat brain identified by [125I]Naja naja siamensis α-toxin binding
350
Brain Research, 131 (1977) 350-355 © Elsevier/North-Holland Biomedical Press
Nicotinic cholinergic receptors in rat brain identified by ['=Sl] Naja naja siamensls a-toxin binding
ROBERT C. SPETH, F. M. CHEN, JON M. LINDSTROM, RONALD M. KOBAYASHI and HENRY I. YAMAMURA* Department of Pharmacology, College of Medicine, University of Arizona Health Science Center, Tucson, Ariz. 85724 and (J. M.L.) The Salk Institute, San Diego, Calif. 92112 and ( R. M.K.) Department of Neurosciences, College of Medicine, University of California at San Diego, La Jolla, Calif 92161 (U.S.A.)
(Accepted April 24th, 1977)
The purified toxins of the elapid snakes, a-bungarotoxin (a-BGT) and a-Naja naja toxin have been used by a large number of investigators to identify, characterize and isolate nicotinic cholinergic receptors (NChR) at neuromuscular junctions and in electric organs of electrophorus and torpedo (see recent reviews in refs.4,6,7,13). More recently a - B G T has been employed to assess N C h R in the mammalian CNS1,3, 5, 9-12,14 16.
In the studies to be reported here, the a-toxin of Naja naja siamensis was employed to characterize and quantitate N C h R in the rat CNS. The a-toxin of Naja naja siamensis (a-NNT) was purified and iodinated with 1251 as described previously 8. The initial specific activity of the 125I-toxin ranged from 60 to 90 Ci/mmole. When the toxin was diluted with non-iodinated toxin, the 125I bound by tissue was decreased by the factor of dilution indicating that the iodinated toxin exhibited the same affinity as the non-iodinated toxin for the toxin binding site. Routine receptor binding assays employed 0.1 ml of rat brain homogenate (100 mg/ml) polytroned on setting no. 5 for 1 min, 0.07 ml of [1251]a-NNT to yield a final concentration of 0.05-1.0 nM, 1.23 ml of 50 m M Na2KPO4 buffer, pH 7.4, containing 1 mg/ml bovine serum albumin, and in alternate sets of three tubes, 0.035 ml of otubocurarine (Calbiochem) to yield a final concentration of 0.1 raM. Incubations were carried out in triplicate at 37 °C for 90 min. Tissue bound toxin was isolated from free toxin by centrifugation in a Beckman microfuge model B, aspiration of the supernatant, one resuspension in ice-cold buffer, a second centrifugation and aspiration of the supernatant. The pellets containing tissue bound 1251 were placed in a Nuclear Chicago gamma counter (Model 4233) and counted at 45-50 ~ efficiency. Specific toxin binding to N C h R was defined as the total amount of toxin bound * To whom correspondence should be addressed at : Department of Pharmacology, Collegeof Medicine University of Arizona, Tucson, Ariz. 85724, U.S.A.
351 6.0
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~
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o
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i
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i
i
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,~1 I. 2 rnln.
.E
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TIME (MINUTES) Fig. 1• Association and dissociation of toxin binding with nicotinic cholinergic receptors. Specific o-tubocurarine displaceable binding to brain homogenate was determined in the presence of 0.5 nM N NT ( × ). At t -- 90 min, 0.1 mM D-turbocurarine was added to alternate sets of 3 tubes to determine the rate at which bound toxin was displaced from specific binding sites (open circles). (a): logarithmic transformation of association rate with least squares regression line. (b) : logarithmic transformation of dissociation rate with least squares regression line.
m i n u s the a m o u n t o f toxin b o u n d in the presence o f 0.1 m M o - t u b o c u r a r i n e (identical values for specific b i n d i n g were o b t a i n e d using a 100-fold excess o f u n l a b e l e d Naja naja siamensis a - t o x i n or a - b u n g a r o t o x i n , o r 0.1 m M nicotine)• Fig. 1 illustrates the time course o f association a n d dissociation o f a-NNT with N C h R . Least square regression lines o b t a i n e d f r o m l o g a r i t h m i c t r a n s f o r m a t i o n s o f a s s o c i a t i o n a n d dissociation time courses yielded half-times o f 11.2 min for a s s o c i a t i o n a n d 26.6 min for dissociation o f the toxin r e c e p t o r complex• These values yielded a dissociation c o n s t a n t (KD) o f 0.2 n M . Fig. 2 illustrates a S c a t c h a r d 17 analysis o f toxin b i n d i n g to whole rat b r a i n yielding a n a p p a r e n t KD value o f 0.73 n M a n d a Mmax value o f 1.83 f m o l e s / m g tissue. Plotting a p p a r e n t KD values versus tissue c o n c e n t r a t i o n to o b t a i n a ' t r u e ' KD for
352 A
,0
20'
E
0
o E ¢3 Z
5 ~BMAx
= 1.83 f m o l e s / m g
0 CO
IZ 0
i
i
i
I
0.5
1.0
1.5
2.0
BOUND/FREE (p.I/lOmg
2.5
31o
tissue)
Fig. 2. Scatchard analysis of toxin binding to rat brain homogenate. Binding was assayed as described in text. an infinitely small tissue concentration z (Fig. 3) yielded 'true' KD value of 0.1 1 n M by least squares analysis.
Pharmacological characterization of toxin binding was determined using various cholinergic ligands (Table I). Both a-bungarotoxin and a-Naja naja siamensis toxin were the most potent inhibitors of labeled toxin binding, with K~ values similar to the Ko for [125I]a-NNT binding obtained in the previous experiments. Nicotinic agonist and antagonist agents yielded K~ values in the micromolar range although the ganglionic nicotinic blocking agents, mecamylamine and hexamethonium, and the depolarizing neuromuscular blocking agent, decamethonium, were far less potent inhibitors. Muscarinic agents also displayed relatively large K~ values. Noncholinergic agents had little or no inhibitory potency at concentrations up to 10 m M . Table II indicates the regional distribution of N C h R in brain. The KD and Bmax
values for each brain area were determined by Scatchard analysis of a-NNT binding. The N C h R appears to be m o s t dense in the hippocampus and hypothalamus and least 0.7 0.6
O
0.5 x~ 0.4 I-Z IJJ
n~ 0.3
0.1
°o
z'5 Jd.o TISSUE (rng)
Fig. 3. Apparent Ko as a function of tissue concentration. Binding was assayed as described in text using 0.05, 0.075 or O.1 ml of brain homogenate.
353 TABLE 1
Pharmacological specificity of [lz51]a-NNT binding to rat brain [125I]a-NNT binding, at 0.3 n M concentration, was assayed as described in the text. Varying amounts of the inhibitors listed below were added and the inhibition of specific, o-tubocurarine displaceable, binding was determined. Hill plots of the inhibition curves yielded IC~0 values. The IC~o values were transformed to K~ values, using a value of 0.16 n M as the KD for [~25I]a-NNT binding, by the following IC50 equation: K~ -- 1 + ligand concentration. KD of ligand Histamine, ouabain and dinitrophenol were inactive in this assay at 10,000/~M concentration.
Blocking agent
Kt (ItM)
a-Bungarotoxin a-Naja naja siamensis toxin Acetylcholine Nicotine o-Tubocurarine Benzoquinonium Dihydro/3-erythroidine Decamethonium Mecamylamine Hexamethonium Atropine Oxotremorine
0.00016 0.00018 2.6 0.23 0.77 9.3 9.7 260 140 370 210 570
TABLE II
Regional distribution of nicotinic binding sites in rat CNS Binding of [125I]a-NNT was assayed as described in text except that varying concentrations of tissue were used so as to maintain approximately equal amounts of a-NNT binding sites in each assay. Scatchard analyses and least square regression analyses were used to obtain apparent KD and Bmax values.
Region
Apparent Ko (riM)
Bm ax (fmoles/mg tissue)
Hippocampus Hypothalamus Midbrain Cerebral cortex Olfactory bulb Brain stem Corpus striatum Spinal cord Cerebellum Whole brain
0.75 0.56 0.58 0.54 0.67 0.48 1.33 0.70 0.50 0.73
3.11 2.69 1.72 1.62 1.40 1.05 0.92 0.73 0.23 1.83
354 dense in the cerebellum. The a p p a r e n t K o values, d e t e r m i n e d with a p p r o x i m a t e l y equal concentrations o f N C h R , did n o t vary significantly in different brain regions. The binding sites for a - N N T a p p e a r to represent the N C h R in the rat brain. The receptor sites show specific, saturable, high affinity binding o f this toxin; they show p h a r m a c o l o g i c a l specificity characteristic o f N C h R a n d a regional distribution in the brain. In addition, the n u m b e r o f binding sites d e t e r m i n e d in this study correlates well with other estimates o f N C h R in brain o b t a i n e d with [125I]a-BGT5,9-12,15,16 a n d [ZH]nicotine is. The N C h R a p p e a r s to be different f r o m the muscarinic cholinergic r e c e p t o r in rat brain because o f the differences in the effects o f muscarinic a n d nicotinic drugs on the receptors 2°, differences in the n u m b e r s o f the two receptors a n d differences in the regional distributions o f these two receptors in the rat b r a i n 21.. The regional distribution o f N C h R shows a reasonable correlation with the regional distributions o f other cholinergic markers~9,2z, 23, with the exception o f the c o r p u s striatum. In view o f the p r e p o n d e r a n c e o f muscarinic receptors in the corpus striatum as well as in o t h e r brain areas one would not necessarily observe a strong correlation between cholinergic m a r k e r s and N C h R . The a u t h o r s wish to express their gratitude to Ms. C a t h y K o u s e n for typing this manuscript. S u p p o r t e d by U S P H S G r a n t s MH-27257, MH-29840 a n d a grant in aid f r o m the C o m m i t t e e to C o m b a t H u n t i n g t o n ' s Disease. H e n r y I. Y a m a m u r a is a recipient o f a Research Scientist D e v e l o p m e n t A w a r d (MH-00095) f r o m the N a t i o n a l Institutes o f M e n t a l Health.
1 Bosmann, H. B., Acetylcholine receptor I. Identification and biochemical characteristics of a cholinergic receptor of guinea pig cortex, J. biol. Chem., 247 (1972) 130-145. 2 Chang, K.-J., Jacobs, S. and Cuatrecasas, P., Quantitative aspects ofhormone-receptor interactions of high affinity. Effect of receptor concentration and measurement of dissociation constants of labeled and unlabeled hormones, Biochim. biophys. Acta (Amst.), 406 (1975) 294-303. 3 De Bias, A. and Mahler, H., Studies on nicotinic acet~lcholine receptors in mammalian brain VI. Isolation of a membrane fraction enriched in receptor function for different neurotransmitters, Biochim. biophys. Res. Commun., 72 (1976) 24-32. 4 Eldefrawi, M. E. and Eldefrawi, A. T., Structure and function of the acetylcholine receptor, Croat. Chem. Acta., 47 (1975) 425-438. 5 Eterovic, V. A. and Bennett, E. L., Nicotinic cholinergic receptor in brain detected by binding of a[3H]bungarotoxin, Biochim. biophys. Acta (Amst.), 362 (1974) 346-355. 6 Fewtrell, C. M. S., The labelling and isolation of neuroreceptors, Neuroscience, 1 (1976) 249-273. 7 Heilbronn, E., Current research on the nature of cholinergic receptors, Croat. Chem. Acta, 47 (1975) 395-408. 8 Lindstr6m, J. M., Lennon, V. A., Seybold, M. E. and Whittingham, S., Experimental autoimmune myasthenia gravis and myasthenia gravis: biochemical and immunochemical aspects, Ann. N. ]I. Acad. Sci., 274 (1976) 254-274. 9 Lowy, J., McGregor, J., Rosenstone, J. and Schmidt, J., Solubilization of an a-bungarotoxinbinding component from rat brain, Biochemistry, 15 (1976) 1522 1527. 10 McQuarrie, C., Salvaterra, P. M., DeBlas, A., Routes, J. and Mahler, H., Studies on nicotinic acetylcholine receptors in mammalian brain preliminary characterization of membrane bound a-bungarotoxin receptors in rat cerebral cortex, J. biol. Chem., 251 (1976) 6335-6339.
355 11 Moore, W. M. and Brady, R. N., Studies of nicotinic acetylcholine receptor protein from rat brain, Biochim. biophys. Acta (Amst.), 444 (1976) 252-260. 12 Polz-Tejera, G., Schmidt, J. and Karten, H. J., Autoradiographic localisation of a-bungarotoxinbinding sites in the central nervous system, Nature (Lond.), 258 (1975) 349-351. 13 Rang, H. P., Acetylcholine receptors, Quart. Rev. Biophys., 7 (1975) 283-399. 14 Salvaterra, P. M. and Mahler, H. R., Nicotinic acetylcholine receptor from rat brain solubilization, partial purification, and characterization, J. biol. Chem., 251 (1976) 6327-6334. 15 Salvaterra, P. M. and Moore, W. J., Binding of [125I]a-bungarotoxin to particulate fractions of rat and guinea pig brain, Biochem. biophys. Res. Commun., 55 (1973) 1311-1318. 16 Salvaterra, P. M., Mahler, H. R. and Moore, W. J., Subcellular and regional distribution ofl25Ilabeled a-bungarotoxin binding in rat brain and its relationship to acetylcholinesterase and choline acetyltransferase, J. bioL Chem., 250 (1975) 6469-6475. 17 Scatchard, G., The attractions of proteins for small molecules and ions, Ann. N.Y. Acad. Sci., 51 (1949) 660-672. 18 Schleifer, L. S. and Eldefrawi, M. E., Identification of the nicotinic and muscarinic acetylcholine receptors in subcellular fractions of mouse brain, Neuropharmacology, 13 (1974) 53-63. 19 Schmidt, D. E., Speth, R. C., Welsch, F. and Schmidt, M. J., The use of microwave radiation in the determination of acetylcholine in the rat brain, Brain Research, 38 (1972) 377-389. 20 Snyder, S. H., Chang, K. J., Kuhar, M. J. and Yamamura, H. I., Biochemical identification of the mammalian muscarinic cholinergic receptor, Fed. Proc., 34 (1975) 1915-1921. 21 Speth, R. C., Chen, F. M., Kobayashi, R., Lindstrom, J. and Yamamura, H. I., Demonstration of brain nicotinic and muscarinic cholinergic receptors, Trans. Amer. Soc. Neurochem., 8 (1977) 121. 22 Yamamura, H. 1. and Snyder, S. H., Muscarinic cholinergic binding in rat brain, Proc. nat. Acad. ScL (Wash.), 71 (1974) 1725-1729. 23 Yamamura, H. I., Kuhar, N. J., Greenberg, D. and Snyder, S. H., Muscarinic cholinergic receptor binding: Regional distribution in monkey brain, Brain Research, 66 (1974) 541-546.
Brain Research, 131 (1977) 350-355 © Elsevier/North-Holland Biomedical Press
Nicotinic cholinergic receptors in rat brain identified by ['=Sl] Naja naja siamensls a-toxin binding
ROBERT C. SPETH, F. M. CHEN, JON M. LINDSTROM, RONALD M. KOBAYASHI and HENRY I. YAMAMURA* Department of Pharmacology, College of Medicine, University of Arizona Health Science Center, Tucson, Ariz. 85724 and (J. M.L.) The Salk Institute, San Diego, Calif. 92112 and ( R. M.K.) Department of Neurosciences, College of Medicine, University of California at San Diego, La Jolla, Calif 92161 (U.S.A.)
(Accepted April 24th, 1977)
The purified toxins of the elapid snakes, a-bungarotoxin (a-BGT) and a-Naja naja toxin have been used by a large number of investigators to identify, characterize and isolate nicotinic cholinergic receptors (NChR) at neuromuscular junctions and in electric organs of electrophorus and torpedo (see recent reviews in refs.4,6,7,13). More recently a - B G T has been employed to assess N C h R in the mammalian CNS1,3, 5, 9-12,14 16.
In the studies to be reported here, the a-toxin of Naja naja siamensis was employed to characterize and quantitate N C h R in the rat CNS. The a-toxin of Naja naja siamensis (a-NNT) was purified and iodinated with 1251 as described previously 8. The initial specific activity of the 125I-toxin ranged from 60 to 90 Ci/mmole. When the toxin was diluted with non-iodinated toxin, the 125I bound by tissue was decreased by the factor of dilution indicating that the iodinated toxin exhibited the same affinity as the non-iodinated toxin for the toxin binding site. Routine receptor binding assays employed 0.1 ml of rat brain homogenate (100 mg/ml) polytroned on setting no. 5 for 1 min, 0.07 ml of [1251]a-NNT to yield a final concentration of 0.05-1.0 nM, 1.23 ml of 50 m M Na2KPO4 buffer, pH 7.4, containing 1 mg/ml bovine serum albumin, and in alternate sets of three tubes, 0.035 ml of otubocurarine (Calbiochem) to yield a final concentration of 0.1 raM. Incubations were carried out in triplicate at 37 °C for 90 min. Tissue bound toxin was isolated from free toxin by centrifugation in a Beckman microfuge model B, aspiration of the supernatant, one resuspension in ice-cold buffer, a second centrifugation and aspiration of the supernatant. The pellets containing tissue bound 1251 were placed in a Nuclear Chicago gamma counter (Model 4233) and counted at 45-50 ~ efficiency. Specific toxin binding to N C h R was defined as the total amount of toxin bound * To whom correspondence should be addressed at : Department of Pharmacology, Collegeof Medicine University of Arizona, Tucson, Ariz. 85724, U.S.A.
351 6.0
xX
/
5.o 6. E
_o 4.0
X x~O
x
~
X
0
x
o
~ ~0 g 0
" 2.0 Z Z 0
__o
=
i
1.0 !
i
i
50
l
I
i
60
TI ME
•0.7: .o) o 0.6
(MINUTES) . b)
,~1 I. 2 rnln.
.E
•
in.
v,v
C0.5 j 0.4
-6.4
"~ 0.3
$.3
t9 0.2
0 .-I
0.1
0 "J.I ~0
I
i 6O
TIME (MINUTES) Fig. 1• Association and dissociation of toxin binding with nicotinic cholinergic receptors. Specific o-tubocurarine displaceable binding to brain homogenate was determined in the presence of 0.5 nM N NT ( × ). At t -- 90 min, 0.1 mM D-turbocurarine was added to alternate sets of 3 tubes to determine the rate at which bound toxin was displaced from specific binding sites (open circles). (a): logarithmic transformation of association rate with least squares regression line. (b) : logarithmic transformation of dissociation rate with least squares regression line.
m i n u s the a m o u n t o f toxin b o u n d in the presence o f 0.1 m M o - t u b o c u r a r i n e (identical values for specific b i n d i n g were o b t a i n e d using a 100-fold excess o f u n l a b e l e d Naja naja siamensis a - t o x i n or a - b u n g a r o t o x i n , o r 0.1 m M nicotine)• Fig. 1 illustrates the time course o f association a n d dissociation o f a-NNT with N C h R . Least square regression lines o b t a i n e d f r o m l o g a r i t h m i c t r a n s f o r m a t i o n s o f a s s o c i a t i o n a n d dissociation time courses yielded half-times o f 11.2 min for a s s o c i a t i o n a n d 26.6 min for dissociation o f the toxin r e c e p t o r complex• These values yielded a dissociation c o n s t a n t (KD) o f 0.2 n M . Fig. 2 illustrates a S c a t c h a r d 17 analysis o f toxin b i n d i n g to whole rat b r a i n yielding a n a p p a r e n t KD value o f 0.73 n M a n d a Mmax value o f 1.83 f m o l e s / m g tissue. Plotting a p p a r e n t KD values versus tissue c o n c e n t r a t i o n to o b t a i n a ' t r u e ' KD for
352 A
,0
20'
E
0
o E ¢3 Z
5 ~BMAx
= 1.83 f m o l e s / m g
0 CO
IZ 0
i
i
i
I
0.5
1.0
1.5
2.0
BOUND/FREE (p.I/lOmg
2.5
31o
tissue)
Fig. 2. Scatchard analysis of toxin binding to rat brain homogenate. Binding was assayed as described in text. an infinitely small tissue concentration z (Fig. 3) yielded 'true' KD value of 0.1 1 n M by least squares analysis.
Pharmacological characterization of toxin binding was determined using various cholinergic ligands (Table I). Both a-bungarotoxin and a-Naja naja siamensis toxin were the most potent inhibitors of labeled toxin binding, with K~ values similar to the Ko for [125I]a-NNT binding obtained in the previous experiments. Nicotinic agonist and antagonist agents yielded K~ values in the micromolar range although the ganglionic nicotinic blocking agents, mecamylamine and hexamethonium, and the depolarizing neuromuscular blocking agent, decamethonium, were far less potent inhibitors. Muscarinic agents also displayed relatively large K~ values. Noncholinergic agents had little or no inhibitory potency at concentrations up to 10 m M . Table II indicates the regional distribution of N C h R in brain. The KD and Bmax
values for each brain area were determined by Scatchard analysis of a-NNT binding. The N C h R appears to be m o s t dense in the hippocampus and hypothalamus and least 0.7 0.6
O
0.5 x~ 0.4 I-Z IJJ
n~ 0.3
0.1
°o
z'5 Jd.o TISSUE (rng)
Fig. 3. Apparent Ko as a function of tissue concentration. Binding was assayed as described in text using 0.05, 0.075 or O.1 ml of brain homogenate.
353 TABLE 1
Pharmacological specificity of [lz51]a-NNT binding to rat brain [125I]a-NNT binding, at 0.3 n M concentration, was assayed as described in the text. Varying amounts of the inhibitors listed below were added and the inhibition of specific, o-tubocurarine displaceable, binding was determined. Hill plots of the inhibition curves yielded IC~0 values. The IC~o values were transformed to K~ values, using a value of 0.16 n M as the KD for [~25I]a-NNT binding, by the following IC50 equation: K~ -- 1 + ligand concentration. KD of ligand Histamine, ouabain and dinitrophenol were inactive in this assay at 10,000/~M concentration.
Blocking agent
Kt (ItM)
a-Bungarotoxin a-Naja naja siamensis toxin Acetylcholine Nicotine o-Tubocurarine Benzoquinonium Dihydro/3-erythroidine Decamethonium Mecamylamine Hexamethonium Atropine Oxotremorine
0.00016 0.00018 2.6 0.23 0.77 9.3 9.7 260 140 370 210 570
TABLE II
Regional distribution of nicotinic binding sites in rat CNS Binding of [125I]a-NNT was assayed as described in text except that varying concentrations of tissue were used so as to maintain approximately equal amounts of a-NNT binding sites in each assay. Scatchard analyses and least square regression analyses were used to obtain apparent KD and Bmax values.
Region
Apparent Ko (riM)
Bm ax (fmoles/mg tissue)
Hippocampus Hypothalamus Midbrain Cerebral cortex Olfactory bulb Brain stem Corpus striatum Spinal cord Cerebellum Whole brain
0.75 0.56 0.58 0.54 0.67 0.48 1.33 0.70 0.50 0.73
3.11 2.69 1.72 1.62 1.40 1.05 0.92 0.73 0.23 1.83
354 dense in the cerebellum. The a p p a r e n t K o values, d e t e r m i n e d with a p p r o x i m a t e l y equal concentrations o f N C h R , did n o t vary significantly in different brain regions. The binding sites for a - N N T a p p e a r to represent the N C h R in the rat brain. The receptor sites show specific, saturable, high affinity binding o f this toxin; they show p h a r m a c o l o g i c a l specificity characteristic o f N C h R a n d a regional distribution in the brain. In addition, the n u m b e r o f binding sites d e t e r m i n e d in this study correlates well with other estimates o f N C h R in brain o b t a i n e d with [125I]a-BGT5,9-12,15,16 a n d [ZH]nicotine is. The N C h R a p p e a r s to be different f r o m the muscarinic cholinergic r e c e p t o r in rat brain because o f the differences in the effects o f muscarinic a n d nicotinic drugs on the receptors 2°, differences in the n u m b e r s o f the two receptors a n d differences in the regional distributions o f these two receptors in the rat b r a i n 21.. The regional distribution o f N C h R shows a reasonable correlation with the regional distributions o f other cholinergic markers~9,2z, 23, with the exception o f the c o r p u s striatum. In view o f the p r e p o n d e r a n c e o f muscarinic receptors in the corpus striatum as well as in o t h e r brain areas one would not necessarily observe a strong correlation between cholinergic m a r k e r s and N C h R . The a u t h o r s wish to express their gratitude to Ms. C a t h y K o u s e n for typing this manuscript. S u p p o r t e d by U S P H S G r a n t s MH-27257, MH-29840 a n d a grant in aid f r o m the C o m m i t t e e to C o m b a t H u n t i n g t o n ' s Disease. H e n r y I. Y a m a m u r a is a recipient o f a Research Scientist D e v e l o p m e n t A w a r d (MH-00095) f r o m the N a t i o n a l Institutes o f M e n t a l Health.
1 Bosmann, H. B., Acetylcholine receptor I. Identification and biochemical characteristics of a cholinergic receptor of guinea pig cortex, J. biol. Chem., 247 (1972) 130-145. 2 Chang, K.-J., Jacobs, S. and Cuatrecasas, P., Quantitative aspects ofhormone-receptor interactions of high affinity. Effect of receptor concentration and measurement of dissociation constants of labeled and unlabeled hormones, Biochim. biophys. Acta (Amst.), 406 (1975) 294-303. 3 De Bias, A. and Mahler, H., Studies on nicotinic acet~lcholine receptors in mammalian brain VI. Isolation of a membrane fraction enriched in receptor function for different neurotransmitters, Biochim. biophys. Res. Commun., 72 (1976) 24-32. 4 Eldefrawi, M. E. and Eldefrawi, A. T., Structure and function of the acetylcholine receptor, Croat. Chem. Acta., 47 (1975) 425-438. 5 Eterovic, V. A. and Bennett, E. L., Nicotinic cholinergic receptor in brain detected by binding of a[3H]bungarotoxin, Biochim. biophys. Acta (Amst.), 362 (1974) 346-355. 6 Fewtrell, C. M. S., The labelling and isolation of neuroreceptors, Neuroscience, 1 (1976) 249-273. 7 Heilbronn, E., Current research on the nature of cholinergic receptors, Croat. Chem. Acta, 47 (1975) 395-408. 8 Lindstr6m, J. M., Lennon, V. A., Seybold, M. E. and Whittingham, S., Experimental autoimmune myasthenia gravis and myasthenia gravis: biochemical and immunochemical aspects, Ann. N. ]I. Acad. Sci., 274 (1976) 254-274. 9 Lowy, J., McGregor, J., Rosenstone, J. and Schmidt, J., Solubilization of an a-bungarotoxinbinding component from rat brain, Biochemistry, 15 (1976) 1522 1527. 10 McQuarrie, C., Salvaterra, P. M., DeBlas, A., Routes, J. and Mahler, H., Studies on nicotinic acetylcholine receptors in mammalian brain preliminary characterization of membrane bound a-bungarotoxin receptors in rat cerebral cortex, J. biol. Chem., 251 (1976) 6335-6339.
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