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Alteration of nicotinic cholinergic agonist binding sites in hippocampus after fimbria transection
Brain Research, 334 (1985) 309-314 Elsevier
309
BRE 10746
Alteration of Nicotinic Cholinergic Agonist Binding Sites in Hippocampus After Fimbria Transection A. LESLIE MORROW 1, REBEKAH LOY2 and IAN CREESE 1 1Department of Neurosciences, University of California, San Diego School of Medicine, La Jolla, CA 92093 and 2Department of Anatomy, University of Rochester School of Medicine, Rochester, NY 14642 (U.S.A.) (Accepted August 21st, 1984) Key words: neuronal nicotinic receptors - - septohippocampal projection - - [3H]acetylcholine - - nicotine treatment
Nicotinic cholinergic agonist binding sites were studied in rat hippocampus by the binding of [3H]acetylcholine in the presence of 1.5 ~M atropine sulfate. Following transection of the fimbria/fornix there was a 49% increase in the binding of [3H]acetylcholine reflecting an increase in the affinity of the receptor binding site from Ka = 18.82 + 3.6 nM in control animals to Ka = 9.06 + 1.2 nM in experimental tissue. Chronic administration of the agonist nicotine (4 mg/kg/day) by osmotic minipumps produced an increase in the binding of 10 nM [3H]acetylcholine after 14 days (49% increase over control) and after 28 days (141% increase over controls). These data are consistent with the suggestion that [3H]acetylcholine labels a nicotinic cholinergic receptor in rat brain. Further they support the notion that some of the termination sites of the septal-cholinergic projection to the hippocampus are nicotinic.
INTRODUCTION
entsS,11,17,23,31,32.
Removal of the septal and diagonal band afferents to the hippocampus depletes acetylcholinesterase and choline acetyltransferase activity in this structure. This and other evidence suggests that this pathway forms the major cholinergic input to the hippocampal formation (see ref. 19). Surprisingly, denervation of these afferents does not alter the binding characteristics of the cholinergic ligands [3H]quinuclidinyl benzilate (QNB) (muscarinic) and [125I]abungarotoxin (nicotinic) (refs. 3, 9, 12, 24, 34; see, however ref. 33). However, it is known that the muscarinic cholinergic sites in brain are capable of substantial up-regulation in response to decreases in agonist stimulation following chronic blockade of receptors with muscarinic antagonists4,21, 22. Thus one explanation for the lack of an increase in hippocampal [3H]QNB binding following cholinergic denervation is that a postsynaptic increase in [3H]QNB binding sites is negated by a simultaneous removal of muscarinic presynaptic receptors on the septal affer-
A n alternative explanation is that the septal cholinergic afferents terminate on a subpopulation of nicotinic cholinergic receptors which are not labeled by [125I]a-bungarotoxin ([125I]a-BTX). The use of [IZ5I]a-BTX as a label for all nicotinic sites has been questioned because: (1) [125I]a-BTX fails to block transmission at many neuronal nicotinic receptors6,7,15,25; (2) iontophoresis of [125I]a-BTX inhibits muscarinic responses (e.g. not induced by nicotine; blocked by atropine) of some pyramidal cells in hippocampus29; and (3) the binding site for [I25I]a-BTX has been localized extrasynaptically in some neuronal tissues such as autonomic ganglia 13. Thus, while there are high affinity [125I]a-BTX sites in hippocampus 2,12,29,30 it is still unclear whether these sites are equivalent to or inclusive of all the functional nicotinic receptors 2,t2,19,29,30. Recently, 3H-agonists have been used to label nicotinic cholinergic sites in the rat brain which have a regional distribution that is different from [125I]aBTX 1,18,26,27,28. In the present study we sought to de-
Correspondence: I. Creese, Department of Neurosciences, University of California, San Diego School of Medicine, La Jolla, CA 92093, U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
310 termine if nicotinic [3H]acetylcholine ([3H]ACh) binding in rat hippocampus would be altered by removal of the septal cholinergic afferents. Further we wished to investigate the regulation of this binding site following chronic treatment with the agonist nicotine. MATERIALS AND METHODS A total of 80 adult female Sprague-Dawley rats (Charles Rivers) was used for these experiments. Transection of the fimbria/fornix was made under pentobarbital anesthesia (45 mg/kg) using a specially constructed tissue knife (3 x 4 mm rectangular blade sharpened on 3 sides) mounted on a stereotaxic frame. Parallel knife cuts were made by placing the blade adjacent to the medial sagittal sinus at AP +0.63 and AP +0.73 from the interaural plane. The blade was then lowered 3.8 mm and moved 2.0 mm medially, 1.0 mm ventrally, 2.0 mm laterally and removed. The rats were kept in colony cages and allowed free access to food and water. Control, non-lesioned rats were from the same litters, sacrificed on the same day and time of day and assayed in parallel with the experimental animals. Eight days after surgery the rats were decapitated and the brains were rapidly removed to chilled saline. Hippocampi were dissected and frozen at -70 °C until assay. The anterior forebrain was placed in 10% formalin and later frozen, sectioned on a cryostat and stained with cresyl violet for lesion reconstruction. Tissues from 4 animals with intact fimbrial fibers at the level of the ventral hippocampal commissure were excluded from analysis. Nicotine tartrate (4 mg/kg/day) was administered for 14 or 28 days via osmotic minipumps (Alza, Palo Alto, CA) implanted subcutaneously. Control rats were implanted with a size matched silastic pellet containing saline. Rats were sacrificed at the end of the final day of treatment, the hippocampus dissected and frozen at -70 °C for 2 weeks when all assays were conducted simultaneously. Radioligand binding assays for [3H]ACh were performed according to the methods of Schwartz et al. zs with minor modifications. Hippocampal tissue was washed twice by centrifugation at 48,000 g with intermediate suspension in fresh buffer composed of 50 mM Tris-HCl, 1.5/aM atropine sulfate (to block mus-
carinic cholinergic receptors), 1.0 mM MgCI:, i2(; mM NaC1, 5 mM KCI and 2 mM CaCI~ (pH 75 at 4 °C). The final suspension of tissue m buffer included 100/aM diisopropylfluorophosphate or l()l) /aM eserine to prevent hydrolysis of the radioligand. [3H]ACh was made in our laboratory' by acetylation of [3H]choline using standard methods and the final purity was checked by high voltage electrophoresis. Our protocol for synthesis yielded greater than 96% purity by incubating 500 ul of [3H]choline (NEN; 80 Ci/mmol) with 50/al ethyl acetate, 10/d acetic anhydride and 10/al triethylamine for 50 rain in polypropylene test tubes. Next 500/d of 95% E T O H was added to convert any remaining anhydride to acetate and subsequently dried under a stream ~ff N:. The [3H]ACh was resuspended in 95% ethanol and stored in the freezer at 4 °C. The assay itself was initiated by adding 300/al of tissue homogenate (final concentration of 20 mg/ml) to polypropylene test tubes containing t00/~1 of radioligand and 100/al of buffer or 100/al of carbachol (100/aM) to determine total and non-specific binding, respectively. All assays were conducted in triplicate for 40 min at 4 °C and terminated by rapid filtration over Whatman GF/C filters soaked m 1% polyethyleneimine. The radioactivity was extracted by shaking for 1 h in Cytoscint (Westchem) and counted at an efficiency of 52%. Specific binding was 65% of total binding at the Ka for [3H]ACh. In order to account for the possible effect of endogenous acetylcholine upon control binding levels, ACh levels were measured in tissue prepared identically for radioligand binding. After the usual washing procedure in the presence of atropine, nicotine (100 /aM) was added to the tissue suspension and allowed to incubate for 40 rain at 0 °C to allow for the dissociation of ACh bound to nicotinic cholinergic receptor sites. The tissue was centrifuged at 40,000 g for 10 min and both the supernatant and pellet were assayed by gas chromatography and mass spectroscopy by the methods of Jenden et al. 14. Residual ACh bound in this pellet was considered to represent neurotransmitter that would be unavailable to compete in a radioligand binding assay while free, unbound ACh in the supernatant would be available to influence control levels of [3H]ACh binding. The binding of [~2H]ct-BTX (161 Ci/mmol) was measured by centrifugation assay in 1.5 ml microfuge
311 TABLE I
Hippocampal nicotinic cholinergic receptor ligand binding 8 days post fimbria/fornix transection Specific binding was determined as described using 10 nM [3H]ACh or 3 nM [125I]R-BTX. Tissue from each animal (n = 14) was divided into two aliquots for simultaneous assay using each ligand. Statistical comparisons were performed using the two-tailed Student's t-test. The control levels of binding for [3H]ACh and [125I]ct-BTX were 0.41 fmol/mg tissue and 1.35 fmol/mg tissue, respectively.
[3H]A Ch specific binding (c.p. ms)
[1251]a-Bungarotoxinspecific binding (c.p. ms)
Control
Control
Lesion
Lesion
(n = 7)
383 + 29.5 %
569 + 29.8 3801 + 196 +48.6 (P < 0.001)
3649+ 110 -4.0 (n.s.)
tubes containing 800 ~1 of tissue (15 mg/ml), 100 ~1 of [125I]ct-BTX (final concentration of 3 nM) and 100 ~1 of buffer or blank drug to d e t e r m i n e non-specific binding. The buffer contained 50 m M Tris HC1, p H 7.7 at 25 °C, 120 m M NaC1 and 2 mg/ml bovine serum albumin. These assays were run in triplicate, incub a t e d at 37 °C for 90 rain and t e r m i n a t e d by centrifugation in a B e c k m a n microfuge for 2 min. T h e super-
taA . k~ X
0
8
natant was aspirated, the tissue r e s u s p e n d e d in 1 ml of cold saline, centrifuged again and the pellets were counted in a g a m m a counter at 79% efficiency. Determinations of non-specific binding were m a d e in the presence of 1 m M D-tubocurarine, and were less than 30% of total binding. RESULTS Eight days after bilateral transection of the timbria/fornix, there is a 48% increase in the specific binding of 10 nM [3H]ACh while there is no change in the binding of 3 nM [125I]a-BTX (Table I). Saturation studies were conducted to d e t e r m i n e if this increase in [3H]ACh binding reflects an increase in receptor capacity or affinity following cholinergic denervation of hippocampus. Fig. 1 illustrates a typical Scatchard plot of the saturation binding d a t a in control and d e n e r v a t e d tissue. The Bmax is unchanged following the surgery; however, there is an increase in binding at the lower ligand concentrations, indicating an increase in the binding affinity of the r e c e p t o r site for [3H]ACh with the K d decreasing from 18.82 + 3.6 nM to 9.06 + 1.2 nM. A potential explanation for an a p p a r e n t increase in affinity is r e m o v a l of e n d o g e n o u s ligand which is depleted by the lesion. This is unlikely since endogenous A C h in control tissue should also be lost by hydrolysis and/or washing during the tissue p r e p a r a tion. H o w e v e r , in o r d e r to examine this possibility we assayed residual A C h following the usual tissue p r e p a r a t i o n and incubation in the absence of radioli-
~
ci3
TABLE II .02
,
.
0
0.3
0.6
Residual hippocampal acetylcholine 8 days post fimbria/fornix transection 0.9
1.2
1.5
BOUND (pmol/g) Fig. 1. Representative Scatchard plot of nicotinic [3H]ACh binding from control hippocampal (E]) and fornix/fimbria transected (A) tissue. Hippocampal tissue from 3 animals was pooled for radioassay in each group. Ligand concentrations ranged from 1.25 to 30 nM. These results were repeated 6 times and no difference in Bmax w a s found between groups. Overall there is a 51.86% decrease in Ka (or increase in affinity) following fimbria transection. Control K d = 18.82 + 3.61 nM; Lesion Ka = 9.06 _+ 1.18 nM; n = 7, P < 0.025, two-tailed t-test.
Membranes were prepared identically as for a binding assay and allowed to incubate for 40 min at 4 °C in the presence of 100 #M nicotine. 1.5/zM atropine sulfate and 100/~M diisopropylfluorophosphate. Following centrifugation, ACh levels were measured in the pellet (trapped) and supernatant (free) and expressed as concentration in a typical binding assay.
Acetylcholine Control Lesion
Trapped (nM)
Free (nM)
67.6 + 4.4 5.88 + 1.6
1.06 ___0.1 0.57 +__0.2
312 gand but in the presence of 100/,m cold nicotine to prevent free ACh from occupancy of nicotinic receptor sites. Table 1I summarizes the data from these experiments. The free residual concentrations of ACh in control and experimental tissue of 1.06 and 0.57 nM are too similar to account for the 50% greater affinity of the [3H]ACh binding sites in the experimental tissue. This endogenous ACh (1 nM) is not sufficient to account for the lower affinity of [3H]ACh in control tissue since the 1Cs0 for acetylcholine in competition with [3H]ACh is 15 nM (data not shown, ref. 28): the theoretical K a in control tissue calculated from the Ka observed but using residual ACh level found in lesion tissue would be 17.83 nM. The levels of trapped ACh are also shown in Table 1I to illustrate the degree of ACh depletion following the lesion. These levels represent ACh bound in vesicles or otherwise unavailable throughout the duration of the routine isotonic binding assay. We found a 90.6% depletion of total ACh following the fimbria transection (n = 5). Treatment with nicotine tartrate also produced a large increase in the binding of 10 nM [3H]ACh (Fig. 2). After 14 days we found a 49% increase in binding which was increased to 141% after 28 days of treatment. Absolute levels of binding were: control
--'• • • (6) Z
200
Z
_J
=o p. z
0 0
lOO CONTROL
* p < .025
* * p < .001
ST) •
2
WEEK
4
WEEK
Fig. 2. Percent control binding of [3H]ACh (10 nM) in rat hippocampal homogenates after continuous treatment with nicotine for 14 or 28 days. Each bar represents the mean -+ S.E.M. normalized with respect to control levels with the number of animals shown in parentheses. Significance was determined by the two-tailed Student's t-test.
0.634 + 0.03 fmol [3H]ACh/mg tissue; i4 day treatment 0.948 + 0.11 fmol/mg tissue; 28 day treatment 1.53 _+ 0.03 fmol/mg tissue. DISCUSSION
The alteration in affinity of nicotinic cholinergic receptor agonist binding sites following cholinergic denervation is consistent with our suggestion that some of the septal afferent terminating cholinergic receptor sites are nicotinic. If [3H]ACh labeled a presynaptic receptor or uptake site on the cholinergic afferents then a decrease in receptor number would have been predicted in the present study. The capacity of nicotinic cholinergic agonist binding sites in hippocampus is low relative to the number of muscarinic cholinergic receptor sites labeled with [3H]QNB (1.3 pmol/g vs 60 pmol/g). This observation and the fact that [3H]QNB binding is also not reduced after fimbria transection suggests that the termination sites are also muscarinic, in part. Our finding of an alteration of [3H]ACh binding sites in response to cholinergic denervation also adds further support to the presumption that [3H]ACh labels a nicotinic cholinergic receptor in mammalian brain. The lack of effect of fimbria/fornix transection on the binding of [~25I]aBTX confirms our preliminary findings u, and adds to a growing body of evidence that this binding site is not identical to sites labeled by [3H]ACh and may not be the functional nicotinic receptor in brain 20. While a change in affinity is an unusual adaptive response to denervation in the case of antagonist radioligandsS, little information is available for agonist radioligands in other systems. For example, agonist binding to receptors coupled to a guanine nucleotide binding protein demonstrate both high and low affinity agonist binding states which are not selectively detected by direct antagonist radioligand binding, Thus it is conceivable that chronic stimulation or depletion of agonists might have effects on receptor affinity measured by [3H]agonist binding which selectively label the high affinity agonist binding state. [3H]ACh binding also demonstrates an atypical response to chronic treatment with the agonist nicotine in that binding is increased as opposed to demonstrating agonist-induced down-regulation 8. While the assays in the present report were done at only one concentration, Schwartz and Kellar 27 recently found that
313 10 day t r e a t m e n t with 4 mg/kg/day nicotine t a r t r a t e increases the Bmax of [3H]ACh binding in the c e r e b r a l cortex by 30%. T h e difference b e t w e e n their result and the larger increases r e p o r t e d h e r e is p r o b a b l y due to the longer t r e a t m e n t p e r i o d and the advantage of continuous administration using i m p l a n t e d osmotic minipumps. I n d e e d we found that 28 days of treatment p r o d u c e d a much greater increase in the binding of [3H]ACh than 14 days of t r e a t m e n t (Fig. 2). A l t h o u g h classified as an agonist by its ability to depolarize the cell, nicotine is a p o t e n t desensitizing agent 10 and this results in b l o c k a d e of further cell firing subsequent to each depolarization. Thus, on continuous administration nicotine m a y act as a functional antagonist and give rise to a c o m p e n s a t o r y nicotinic cholinergic r e c e p t o r up-regulation. It would be of special interest, then, to examine [3H]ACh binding after chronic t r e a t m e n t with an antagonist drug which lacks any agonist activity. Surprisingly, an an-
REFEREI'~CES 1 Abood, L. G., Grassi, S. and Costanza, M., Binding of optically pure (-)3H-nicotine to rat brain membranes, FEBS Lett., 157 (1983) 147-149. 2 Ben-Barak, J. and Dudai, Y., Cholinergic binding sites in rat hippocampal formation: properties and ontogenesis, Brain Research, 166 (1979) 245-257. 3 Ben-Barak, J. and Dudai, Y., Early septal lesion: effect on development of the cholinergic system in rat hippocampus, Brain Research, 185 (1980) 323-334. 4 Ben-Barak, J., Gazit, H., Silman, I. and Dudai, Y., In vivo modulation of the number of muscarinic receptors in rat brain by cholinergic ligands, Europ. J. Pharmacol., 74 (1981) 73-81. 5 Bourdois, P. S., Mitchell, J. F., Somogyi, G. T. and Szerb, J. C., The output per stimulus of acetylcholine from cerebral cortical slices in the presence or absence of cholinesterase inhibition, Brit. J. Pharmacol., 52 (1974) 509-517. 6 Brown, D. A. and Fumagalli, L., Dissociation of a-bungarotoxin binding and receptor block in the rat superior cervical ganglion, Brain Research, 129 (1977) 165-168. 7 Carbonetto, S, T., Fambrough, D. M. and Muller, K. J., Nonequivalence of a-bungarotoxin receptors and acetylcholine receptors in chick sympathetic neurons, Proc. nat. Acad. Sci. U.S.A., 75 (1978) 1016-1020. 8 Creese, I. and Sibley, D. R., Receptor adaptations to centrally acting drugs, Ann. Rev. Pharmacol. Toxicol., 21 (1980) 357-391. 9 Dudai, Y. and Segat, M., a-Bungarotoxin binding sites in rat hippocampus: localization in post-synaptic cells, Brain Research, 154 (1978) 167-171. 10 Ginsborg, B. L. and Guerrero, S., On the action of depolarizing drugs on sympathetic ganglion cells of the frog, J. Physiol. (Lond.), 172 (1964) 189-206. 11 Hounsgaard, J., Presynaptic inhibitory action of acetylcho-
tagonist drug of high n a n o m o l a r (e.g. < 20 nM) affinity has yet to be identified for the [3H]ACh binding site. ACKNOWLEDGEMENTS W e wish to thank Dr. D o n a l d J e n d e n and Kathleen Rice for their assistance with the acetylcholine assays. Purified [125I]a-BTX was kindly p r o v i d e d by Dr. Darwin Berg. A n d r e w Chen is t h a n k e d for his expert technical assistance and we are grateful to Dolores Taitano for word-processing. This work was s u p p o r t e d by PHS NS17860 (I.C. and R . L . ) , NS20288 ( R . L . ) , MH17691 (Dr. Jenden) and by the March of Dimes 1-822 (I.C. and R . L . ) . I.C. holds a Research Scientist Development Award (MH00316). A . L . M . Is a National Institute of Mental Health Predoctoral Fellow (MH08898).
line in area CA1 of the hippocampus, Exp. Neurol., 62 (1978) 787-797. 12 Hunt, S. and Schmidt, J., The relationship of a-bungarotoxin binding activity and cholinergic termination within the rat hippocampus, Neuroscience, 4 (1979) 585-592. 13 Jacob, M. H. and Berg, D. K., a-Bungarotoxin binds to developing ciliary ganglion neurons but not at synapses, Soc. Neurosci. Abstr., 7 (1982) 702, 14 Jenden, D. J., Roch, M. and Booth, R. A., Simultaneous measurement of endogenous and deuterium-labelled tracer variants of choline and acetylcholine in subpicomolar quantities by gas chromatography and mass spectroscopy, Anal. Biochem., 55 (1973) 438-448. 15 Kouvelas, E. D., Dichter, M. A. and Greene, L. A., Chick sympathetic neurons develop receptors for a-bungarotoxin in vitro but the toxin does not block nicotinic receptors, Brain Research, 154 (1978) 83-98. 16 Loy, R., Morrow, A. L, and Creese, I., Cholinergic denervation induces adrenergic receptor increases: correlation with sympathetic axon sprouting in hippocampus, Birth Defects: Orig. Art. Ser., 19 (1983) 409-415. 17 Marchi, M., Paudice, P. and Raiteri, M., Presynaptic autoreceptors controlling acetylcholine release in synaptosomes from rat hippocampus, Proc. Brit. Pharmacol. Soc., (1981) 220P. 18 Marks, M. J. and Collins, A. C., Characterization of nicotine binding in mouse brain and comparison with the binding of a-bungarotoxin and quinuclidinyl benzilate, Molec. PharmacoL, 22 (1982) 554-564. 19 Milner, T. A., Loy, R. and Amaral, D. G., An anatomical study of the development of the septo-hippocampal projection in the rat, Develop. Brain Res., 8 (1983) 343-371. 20 Morley, B. J., Kemp, G. E. and Salvaterra, P., a-Bungarotoxin binding sites in the CNS, Life Sci., 24 (1979) 859-872. 21 Morrow, A. L., Wurtz, C. W., Loy, R. and Creese, I., Cholinergic denervation and blockade increase adrenergic
314 binding in rat brain, Soc. Neurosci. A bstr., 7 (1981) 151. 22 Morrow, A. L., Loy, R. and Creese, 1., Septal deafferentation increases hippocampal adrenergic receptors: correlation with sympathetic axon sprouting. Proc. nat. Aead. Sci. U.S.A., 80 (1983) 6718-6722. 23 Nordstrom, O. and Bartfai, T., Muscarinic autoreceptor regulates acetylcholine release in rat hippocampus: in vitro evidence, Actaphysiol. scand., 108 (1980) 347-353. 24 Overstreet, D. H., Speth, R. C., Hruska, R. E., Ehlert, F.. Dumont, E. Y. and Yamamura, H. I., Failure of septal lesions to alter muscarinic or benzodiazepine binding sites in hippocampus of rat brain, Brain Research, 195 (1980) 203-207. 25 Patrick, J. and Stallcup, W. B., Immunological distinction between acetylcholine receptor and the ct-bungarotoxin binding component on sympathetic neurons, Proc. nat. Acad. Sci. U.S.A., 74 (1977) 4689-4692. 26 Romano, C. and Goldstein, A., Stereospecific nicotine receptors on rat brain membranes, Science, 210 (1980) 647- 649. 27 Schwartz, R. D. and Kellar, K. J., Nicotinic cholinergic receptor binding sites in the brain: regulation in vivo, Science, 220 (1983) 214-216. 28 Schwartz, R. D., McGee, R. and Kellar, K. J., Nicotinic
29
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31
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33
34
cholinergic receptors labelled by 3H-acetvlcholine m ~ brain, Molec. Pharmacol., 22 (1982) 56- 62 Segal, M., The acetylcholine receptor in the ~at hippocam~ pus: nicotinic, muscarinic or both? Neuropharmacolo~v. 17' (1978) 619-623. Segal, M., Dudai, Y. and Amsterdam, A., Distribution or' an a-bungarotoxin-binding cholinergic nicotinic receptors in rat brain, Brain Research, 148 (1978) 105- I i9. Szerb, J. C., Characterization of presynaptic muscarinic receptors in central cholinergic neurons, h: D. J. Jenden (Ed.), Cholinergic Mechanisms and Psychopharmacology, Plenum Press, New York, 1977. pp. 49-60. Szerb, J. C.. Hadhazy, P. and Dudar, J 1_). Release oi [3H]ACh from rat hippocampal slices: effect of septal lesion and of graded concentrations of muscarinic agonists and antagonists, Brain Research, 128 (1977) 285-29I. Westlind, A., Grynfarb, M.. ttedlund, B.. Bartfai, T. and Fuxe, L., Muscarinic supersensitivity induced by septal lesion or chronic atropine treatment, Brain Research, 225 (1981) 131-141. Yamamura, H. I. and Snyder, S. H., Postsynaptic localization of muscarinic cholinergic receptor binding in rat hippocampus, Brain Research, 78 (1974) 320-326.
309
BRE 10746
Alteration of Nicotinic Cholinergic Agonist Binding Sites in Hippocampus After Fimbria Transection A. LESLIE MORROW 1, REBEKAH LOY2 and IAN CREESE 1 1Department of Neurosciences, University of California, San Diego School of Medicine, La Jolla, CA 92093 and 2Department of Anatomy, University of Rochester School of Medicine, Rochester, NY 14642 (U.S.A.) (Accepted August 21st, 1984) Key words: neuronal nicotinic receptors - - septohippocampal projection - - [3H]acetylcholine - - nicotine treatment
Nicotinic cholinergic agonist binding sites were studied in rat hippocampus by the binding of [3H]acetylcholine in the presence of 1.5 ~M atropine sulfate. Following transection of the fimbria/fornix there was a 49% increase in the binding of [3H]acetylcholine reflecting an increase in the affinity of the receptor binding site from Ka = 18.82 + 3.6 nM in control animals to Ka = 9.06 + 1.2 nM in experimental tissue. Chronic administration of the agonist nicotine (4 mg/kg/day) by osmotic minipumps produced an increase in the binding of 10 nM [3H]acetylcholine after 14 days (49% increase over control) and after 28 days (141% increase over controls). These data are consistent with the suggestion that [3H]acetylcholine labels a nicotinic cholinergic receptor in rat brain. Further they support the notion that some of the termination sites of the septal-cholinergic projection to the hippocampus are nicotinic.
INTRODUCTION
entsS,11,17,23,31,32.
Removal of the septal and diagonal band afferents to the hippocampus depletes acetylcholinesterase and choline acetyltransferase activity in this structure. This and other evidence suggests that this pathway forms the major cholinergic input to the hippocampal formation (see ref. 19). Surprisingly, denervation of these afferents does not alter the binding characteristics of the cholinergic ligands [3H]quinuclidinyl benzilate (QNB) (muscarinic) and [125I]abungarotoxin (nicotinic) (refs. 3, 9, 12, 24, 34; see, however ref. 33). However, it is known that the muscarinic cholinergic sites in brain are capable of substantial up-regulation in response to decreases in agonist stimulation following chronic blockade of receptors with muscarinic antagonists4,21, 22. Thus one explanation for the lack of an increase in hippocampal [3H]QNB binding following cholinergic denervation is that a postsynaptic increase in [3H]QNB binding sites is negated by a simultaneous removal of muscarinic presynaptic receptors on the septal affer-
A n alternative explanation is that the septal cholinergic afferents terminate on a subpopulation of nicotinic cholinergic receptors which are not labeled by [125I]a-bungarotoxin ([125I]a-BTX). The use of [IZ5I]a-BTX as a label for all nicotinic sites has been questioned because: (1) [125I]a-BTX fails to block transmission at many neuronal nicotinic receptors6,7,15,25; (2) iontophoresis of [125I]a-BTX inhibits muscarinic responses (e.g. not induced by nicotine; blocked by atropine) of some pyramidal cells in hippocampus29; and (3) the binding site for [I25I]a-BTX has been localized extrasynaptically in some neuronal tissues such as autonomic ganglia 13. Thus, while there are high affinity [125I]a-BTX sites in hippocampus 2,12,29,30 it is still unclear whether these sites are equivalent to or inclusive of all the functional nicotinic receptors 2,t2,19,29,30. Recently, 3H-agonists have been used to label nicotinic cholinergic sites in the rat brain which have a regional distribution that is different from [125I]aBTX 1,18,26,27,28. In the present study we sought to de-
Correspondence: I. Creese, Department of Neurosciences, University of California, San Diego School of Medicine, La Jolla, CA 92093, U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
310 termine if nicotinic [3H]acetylcholine ([3H]ACh) binding in rat hippocampus would be altered by removal of the septal cholinergic afferents. Further we wished to investigate the regulation of this binding site following chronic treatment with the agonist nicotine. MATERIALS AND METHODS A total of 80 adult female Sprague-Dawley rats (Charles Rivers) was used for these experiments. Transection of the fimbria/fornix was made under pentobarbital anesthesia (45 mg/kg) using a specially constructed tissue knife (3 x 4 mm rectangular blade sharpened on 3 sides) mounted on a stereotaxic frame. Parallel knife cuts were made by placing the blade adjacent to the medial sagittal sinus at AP +0.63 and AP +0.73 from the interaural plane. The blade was then lowered 3.8 mm and moved 2.0 mm medially, 1.0 mm ventrally, 2.0 mm laterally and removed. The rats were kept in colony cages and allowed free access to food and water. Control, non-lesioned rats were from the same litters, sacrificed on the same day and time of day and assayed in parallel with the experimental animals. Eight days after surgery the rats were decapitated and the brains were rapidly removed to chilled saline. Hippocampi were dissected and frozen at -70 °C until assay. The anterior forebrain was placed in 10% formalin and later frozen, sectioned on a cryostat and stained with cresyl violet for lesion reconstruction. Tissues from 4 animals with intact fimbrial fibers at the level of the ventral hippocampal commissure were excluded from analysis. Nicotine tartrate (4 mg/kg/day) was administered for 14 or 28 days via osmotic minipumps (Alza, Palo Alto, CA) implanted subcutaneously. Control rats were implanted with a size matched silastic pellet containing saline. Rats were sacrificed at the end of the final day of treatment, the hippocampus dissected and frozen at -70 °C for 2 weeks when all assays were conducted simultaneously. Radioligand binding assays for [3H]ACh were performed according to the methods of Schwartz et al. zs with minor modifications. Hippocampal tissue was washed twice by centrifugation at 48,000 g with intermediate suspension in fresh buffer composed of 50 mM Tris-HCl, 1.5/aM atropine sulfate (to block mus-
carinic cholinergic receptors), 1.0 mM MgCI:, i2(; mM NaC1, 5 mM KCI and 2 mM CaCI~ (pH 75 at 4 °C). The final suspension of tissue m buffer included 100/aM diisopropylfluorophosphate or l()l) /aM eserine to prevent hydrolysis of the radioligand. [3H]ACh was made in our laboratory' by acetylation of [3H]choline using standard methods and the final purity was checked by high voltage electrophoresis. Our protocol for synthesis yielded greater than 96% purity by incubating 500 ul of [3H]choline (NEN; 80 Ci/mmol) with 50/al ethyl acetate, 10/d acetic anhydride and 10/al triethylamine for 50 rain in polypropylene test tubes. Next 500/d of 95% E T O H was added to convert any remaining anhydride to acetate and subsequently dried under a stream ~ff N:. The [3H]ACh was resuspended in 95% ethanol and stored in the freezer at 4 °C. The assay itself was initiated by adding 300/al of tissue homogenate (final concentration of 20 mg/ml) to polypropylene test tubes containing t00/~1 of radioligand and 100/al of buffer or 100/al of carbachol (100/aM) to determine total and non-specific binding, respectively. All assays were conducted in triplicate for 40 min at 4 °C and terminated by rapid filtration over Whatman GF/C filters soaked m 1% polyethyleneimine. The radioactivity was extracted by shaking for 1 h in Cytoscint (Westchem) and counted at an efficiency of 52%. Specific binding was 65% of total binding at the Ka for [3H]ACh. In order to account for the possible effect of endogenous acetylcholine upon control binding levels, ACh levels were measured in tissue prepared identically for radioligand binding. After the usual washing procedure in the presence of atropine, nicotine (100 /aM) was added to the tissue suspension and allowed to incubate for 40 rain at 0 °C to allow for the dissociation of ACh bound to nicotinic cholinergic receptor sites. The tissue was centrifuged at 40,000 g for 10 min and both the supernatant and pellet were assayed by gas chromatography and mass spectroscopy by the methods of Jenden et al. 14. Residual ACh bound in this pellet was considered to represent neurotransmitter that would be unavailable to compete in a radioligand binding assay while free, unbound ACh in the supernatant would be available to influence control levels of [3H]ACh binding. The binding of [~2H]ct-BTX (161 Ci/mmol) was measured by centrifugation assay in 1.5 ml microfuge
311 TABLE I
Hippocampal nicotinic cholinergic receptor ligand binding 8 days post fimbria/fornix transection Specific binding was determined as described using 10 nM [3H]ACh or 3 nM [125I]R-BTX. Tissue from each animal (n = 14) was divided into two aliquots for simultaneous assay using each ligand. Statistical comparisons were performed using the two-tailed Student's t-test. The control levels of binding for [3H]ACh and [125I]ct-BTX were 0.41 fmol/mg tissue and 1.35 fmol/mg tissue, respectively.
[3H]A Ch specific binding (c.p. ms)
[1251]a-Bungarotoxinspecific binding (c.p. ms)
Control
Control
Lesion
Lesion
(n = 7)
383 + 29.5 %
569 + 29.8 3801 + 196 +48.6 (P < 0.001)
3649+ 110 -4.0 (n.s.)
tubes containing 800 ~1 of tissue (15 mg/ml), 100 ~1 of [125I]ct-BTX (final concentration of 3 nM) and 100 ~1 of buffer or blank drug to d e t e r m i n e non-specific binding. The buffer contained 50 m M Tris HC1, p H 7.7 at 25 °C, 120 m M NaC1 and 2 mg/ml bovine serum albumin. These assays were run in triplicate, incub a t e d at 37 °C for 90 rain and t e r m i n a t e d by centrifugation in a B e c k m a n microfuge for 2 min. T h e super-
taA . k~ X
0
8
natant was aspirated, the tissue r e s u s p e n d e d in 1 ml of cold saline, centrifuged again and the pellets were counted in a g a m m a counter at 79% efficiency. Determinations of non-specific binding were m a d e in the presence of 1 m M D-tubocurarine, and were less than 30% of total binding. RESULTS Eight days after bilateral transection of the timbria/fornix, there is a 48% increase in the specific binding of 10 nM [3H]ACh while there is no change in the binding of 3 nM [125I]a-BTX (Table I). Saturation studies were conducted to d e t e r m i n e if this increase in [3H]ACh binding reflects an increase in receptor capacity or affinity following cholinergic denervation of hippocampus. Fig. 1 illustrates a typical Scatchard plot of the saturation binding d a t a in control and d e n e r v a t e d tissue. The Bmax is unchanged following the surgery; however, there is an increase in binding at the lower ligand concentrations, indicating an increase in the binding affinity of the r e c e p t o r site for [3H]ACh with the K d decreasing from 18.82 + 3.6 nM to 9.06 + 1.2 nM. A potential explanation for an a p p a r e n t increase in affinity is r e m o v a l of e n d o g e n o u s ligand which is depleted by the lesion. This is unlikely since endogenous A C h in control tissue should also be lost by hydrolysis and/or washing during the tissue p r e p a r a tion. H o w e v e r , in o r d e r to examine this possibility we assayed residual A C h following the usual tissue p r e p a r a t i o n and incubation in the absence of radioli-
~
ci3
TABLE II .02
,
.
0
0.3
0.6
Residual hippocampal acetylcholine 8 days post fimbria/fornix transection 0.9
1.2
1.5
BOUND (pmol/g) Fig. 1. Representative Scatchard plot of nicotinic [3H]ACh binding from control hippocampal (E]) and fornix/fimbria transected (A) tissue. Hippocampal tissue from 3 animals was pooled for radioassay in each group. Ligand concentrations ranged from 1.25 to 30 nM. These results were repeated 6 times and no difference in Bmax w a s found between groups. Overall there is a 51.86% decrease in Ka (or increase in affinity) following fimbria transection. Control K d = 18.82 + 3.61 nM; Lesion Ka = 9.06 _+ 1.18 nM; n = 7, P < 0.025, two-tailed t-test.
Membranes were prepared identically as for a binding assay and allowed to incubate for 40 min at 4 °C in the presence of 100 #M nicotine. 1.5/zM atropine sulfate and 100/~M diisopropylfluorophosphate. Following centrifugation, ACh levels were measured in the pellet (trapped) and supernatant (free) and expressed as concentration in a typical binding assay.
Acetylcholine Control Lesion
Trapped (nM)
Free (nM)
67.6 + 4.4 5.88 + 1.6
1.06 ___0.1 0.57 +__0.2
312 gand but in the presence of 100/,m cold nicotine to prevent free ACh from occupancy of nicotinic receptor sites. Table 1I summarizes the data from these experiments. The free residual concentrations of ACh in control and experimental tissue of 1.06 and 0.57 nM are too similar to account for the 50% greater affinity of the [3H]ACh binding sites in the experimental tissue. This endogenous ACh (1 nM) is not sufficient to account for the lower affinity of [3H]ACh in control tissue since the 1Cs0 for acetylcholine in competition with [3H]ACh is 15 nM (data not shown, ref. 28): the theoretical K a in control tissue calculated from the Ka observed but using residual ACh level found in lesion tissue would be 17.83 nM. The levels of trapped ACh are also shown in Table 1I to illustrate the degree of ACh depletion following the lesion. These levels represent ACh bound in vesicles or otherwise unavailable throughout the duration of the routine isotonic binding assay. We found a 90.6% depletion of total ACh following the fimbria transection (n = 5). Treatment with nicotine tartrate also produced a large increase in the binding of 10 nM [3H]ACh (Fig. 2). After 14 days we found a 49% increase in binding which was increased to 141% after 28 days of treatment. Absolute levels of binding were: control
--'• • • (6) Z
200
Z
_J
=o p. z
0 0
lOO CONTROL
* p < .025
* * p < .001
ST) •
2
WEEK
4
WEEK
Fig. 2. Percent control binding of [3H]ACh (10 nM) in rat hippocampal homogenates after continuous treatment with nicotine for 14 or 28 days. Each bar represents the mean -+ S.E.M. normalized with respect to control levels with the number of animals shown in parentheses. Significance was determined by the two-tailed Student's t-test.
0.634 + 0.03 fmol [3H]ACh/mg tissue; i4 day treatment 0.948 + 0.11 fmol/mg tissue; 28 day treatment 1.53 _+ 0.03 fmol/mg tissue. DISCUSSION
The alteration in affinity of nicotinic cholinergic receptor agonist binding sites following cholinergic denervation is consistent with our suggestion that some of the septal afferent terminating cholinergic receptor sites are nicotinic. If [3H]ACh labeled a presynaptic receptor or uptake site on the cholinergic afferents then a decrease in receptor number would have been predicted in the present study. The capacity of nicotinic cholinergic agonist binding sites in hippocampus is low relative to the number of muscarinic cholinergic receptor sites labeled with [3H]QNB (1.3 pmol/g vs 60 pmol/g). This observation and the fact that [3H]QNB binding is also not reduced after fimbria transection suggests that the termination sites are also muscarinic, in part. Our finding of an alteration of [3H]ACh binding sites in response to cholinergic denervation also adds further support to the presumption that [3H]ACh labels a nicotinic cholinergic receptor in mammalian brain. The lack of effect of fimbria/fornix transection on the binding of [~25I]aBTX confirms our preliminary findings u, and adds to a growing body of evidence that this binding site is not identical to sites labeled by [3H]ACh and may not be the functional nicotinic receptor in brain 20. While a change in affinity is an unusual adaptive response to denervation in the case of antagonist radioligandsS, little information is available for agonist radioligands in other systems. For example, agonist binding to receptors coupled to a guanine nucleotide binding protein demonstrate both high and low affinity agonist binding states which are not selectively detected by direct antagonist radioligand binding, Thus it is conceivable that chronic stimulation or depletion of agonists might have effects on receptor affinity measured by [3H]agonist binding which selectively label the high affinity agonist binding state. [3H]ACh binding also demonstrates an atypical response to chronic treatment with the agonist nicotine in that binding is increased as opposed to demonstrating agonist-induced down-regulation 8. While the assays in the present report were done at only one concentration, Schwartz and Kellar 27 recently found that
313 10 day t r e a t m e n t with 4 mg/kg/day nicotine t a r t r a t e increases the Bmax of [3H]ACh binding in the c e r e b r a l cortex by 30%. T h e difference b e t w e e n their result and the larger increases r e p o r t e d h e r e is p r o b a b l y due to the longer t r e a t m e n t p e r i o d and the advantage of continuous administration using i m p l a n t e d osmotic minipumps. I n d e e d we found that 28 days of treatment p r o d u c e d a much greater increase in the binding of [3H]ACh than 14 days of t r e a t m e n t (Fig. 2). A l t h o u g h classified as an agonist by its ability to depolarize the cell, nicotine is a p o t e n t desensitizing agent 10 and this results in b l o c k a d e of further cell firing subsequent to each depolarization. Thus, on continuous administration nicotine m a y act as a functional antagonist and give rise to a c o m p e n s a t o r y nicotinic cholinergic r e c e p t o r up-regulation. It would be of special interest, then, to examine [3H]ACh binding after chronic t r e a t m e n t with an antagonist drug which lacks any agonist activity. Surprisingly, an an-
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