Distribution of α-bungarotoxin binding sites in the central nervous system and peripheral organs of the rat

To .rrron, 1978, Vol. 16, pp . 245-211 . Pergattton Prcae . Printed in Great Britain

DISTRIBUTION OF a-BUNGAROTOXIN BINDING SITES IN THE CENTRAL NERVOUS SYSTEM AND PERIPHERAL ORGANS OF THE RAT NISSON SCHECHTER,* INDHIRA C. HANDY,t LEO PEZZEMENTI,t and .TAKOB ScHhnnTt$ 'Long Island Research Institute and Department of Psychiatry and Behavioral Science, State University of New York at Stony Brook, Stony Brook, N.Y. 11794, U.S.A., tDeparhnent of Biochemistry, State University of New York at Stony Brook, Stony Brook, N.Y .11794, U.S.A. (Accepted for publication 18 August 1977) L. PezzeMelvrt and J. Scx~r . Distribution of a-bungarotoxin binding sites in the central nervous system and peripheral organs of the rat . Toxfcon 16, 245-251, 1978.--Concentrntions of a-bungarotoxin binding sites were determined biochemically in various regions of the central nervous system and several peripheral tissues of the rat . Marked regional differences were found within the brain. No activity was detected in the caudate nucleus and little in the cerebellum; highest levels were observed in the colliculus inferior and superior and the hippocampus and intermediate densities were found in other areas . Outside of the central nervous system, binding activity was significant only in skeletal muscle, autonomic ganglia and adrenal gland; in addition, a-bungarotoxin retxptora were found in sensory ganglia (Gasserian; dorsal root). Tâe findings of this survey suggest that a-bungarotoxin is specific for nicotinic acetylcholine receptors regardless of their location within the organism, and that these receptors are not necessarily synaptic constituents. N. SCHECHIER, I. C. HANDY,

INTRODUCTION a-BUNGAROTOXIN (aBuTX) and related snake neurotoxins bind with high affinity and specificity to acetylcholine receptors in muscle and electric organ (Hall, 1972 ; CHANGEUX, 1975). Binding sites for aBuTX have also been described in chick sympathetic ganglion neurones (GREENS et al., 1973 ; GREENS, 1976) and in the central nervous system, including the retina, of various species (ET -EROVI~ and BENNI?z-r, 1974 ; $AIVATERRA et al., 1975 ; POIZ-TEJERA et al., 1975 ; LowY et al., 1976; SAIVATERRA and MAHIER, 1976; VOGEL and NIRENBERO, 1976 ; YAZULLA arid SCHMIDT, 1976 ; SETO et al., 1977) . It is plausible to assume that these neuronal toxin receptors are likewise nicotinic acetylcholine receptors, a notion supported by several observations : central toxin receptors are membrane-bound and resemble peripheral nicotinic receptors with respect to detergent solubility, isolectric point, and radius of gyration (LovrY et al., 1976; SALVATERRA and MAHLER, 1976 ; SETO et aL, 1977). An analysis of the effect of neuroactive drugs on toxin binding rate has shown that classical nicotinic drugs bind with high affinity, while muscarinic and non-cholinergic ligands interact but weakly with these receptors (ScHMIDr, 1977) . Toxin receptors are enriched in preparations of nerve endings (SAIVA7ERRA et al., 1975) and especially in purified synaptic membranes (BARTFAI et al., 1976; DE BIAS and MAI-II.ER, 1976) . Finally, autoradiographic studies of fish, reptilian and avian retina at the light-microscopic level have shown that receptor sites are confined to known synaptic regions (VOGEL and NIRSNBERIi, 1976 ; YAZULLA and SCHMIDT, 1976) . ~To whom correspondence should be mailed . 245

24 6

N. SCHECHTER, I. C. HANDY, L. PEZZEMENTI and J. SCHMmT

Unfortunately, however, it has not been possible to demonstrate an in vivo effect of on cholinergic synapses in the central nervous system or between neurones. Attempts to block nicotinic receptors in the spinal cord of the frog (MII .EDI and SzczHPAx1Ax, 1975) and cat (DUGGAN et al., 197 and in sympathetic ganglia of the cat (CHOU and Lam, 1969) and rat (BROwx and FuMACALIa, 1977) by application of aBuTX or Naja raja siamensis and Naja naja afro postsynaptic neurotoxins have failed. Mesl .AlvD and Aa~s (1976) observed a blocking effect of B. multicinctus venom in the rabbit retina, but it is not clear if this was due to aBuTX. These observations do not rule out the possibility that binding sites for aBuTX are related in some fashion to neurotransmitter receptors, but we certainly cannot take it for granted any longer that aBuTX receptors in the CNS are neurotransmitter receptors. One approach to the problem of receptor identity which to our knowledge has not yet been undertaken in a systematic manner is to survey toxin receptor distribution in the whole body . Such an inventory could answer two questions which bear on the problem of toxin specificity (1) Is toxin binding observed only in tissues that are known to contain nicotinic acetylcholine receptors? (2) Is the localization of toxin receptors within the central nervous system compatible with what it known about the occurrence of nicotinic synapses? In this communication we present a survey of receptor densities in various tissues. The results are in reasonable agreement with pharmacological predictions, hereby supporting the view that aBuTX binds to nicotinic acetylcholine receptors not only in muscle fibers but also in nerve cells. aBuTX

MATERIALS AND METHODS The purification and iodination of aBuTX were carried out as described previously (Low+v et al., 197 . Burrgartu mrdticirrctus venom was purchased from Miami Serpentarium (Florida) aced fractionated by chromatography on carboxymethyl cellulose (Whatman CM 52) and Sephadex G-SO (Pharmacia). T'he pun a-toxin was iodinated using the chloramine T procedure and the labeled product separated from unlabeled toxin by chromatography on carboxymethyl cellulose. The specific activity of 1"I-aBuTX was 10' Ci/mole or lower, depending on the age of the preparation . The iodinated toxin was found to retain its biological activity (defined as the ability to bind to Torpedo californica electric organ receptors) over more than two 1"I half lives (i.o . more than four months). Sprague-Dawley rata, weighing 120-300 g, were sacrificed by decapitation, the desiredtissue removed and homogenized in 9 vol of 100 mM sodium phosphate ; 0"4mM phenyhnethyl aulfonyl fluoride ; l mM sodium EDTA ; 0"02 ~ sodium azide; pH 7"4, using a glass tissue grinder. when solubt7ization of the toxin receptor was desired 0"1 volume of 10~ Triton X-100 was added to the homogenate, and, after stirring for ono hour at room temperature, the detergent extract obtained by centrifuging the sample for 30 min at 100,000 x g. Preparations were assayed for aBuTX binding activity without delay. Toxin binding to particulate preparations was analyzed by means of a centrifuge assay whose details have been given in a previous paper (ScFn~T,1977). Briefly, homogenates representing about 10 mg of wet tissue were incubated with 100 femtomoles of 1~IßBuTX, in 1"Sml Eppendorf centrifuge tubes, in a total volume of 0"5 m1,10 mM sodium phosphate, pH 7"4. After 3 hr at room temperature, 0~8 ml of 0"2 M sodium chloride was added, free toxin removed by three cycles of centrifugation (in an Eppendorf model 3200 centrifuge), aspiration, and resuapension, and bound radioactivity measured by digesting the washed pellet in 0"2 ml Protasol (New England Nuclear) and counting it, after addition of 12 ml of 0"4~ Permablend III (Packard) in toluene, in a liquid scintillation spectrometer . This assay slightly (i .e ., by about 10 %~ underestimates receptor levels in brain particulates as some membranes do not sediment under the centrifugation conditions employed and a small fraction of the receptor-toxin complex dissociates during the wash procedure. No attempt was made to determine the extent of such losses for other tissues. Binding activity of skeletal muscle was measured using the DEAE~ellulose disc technique ($CHII®T & RAFIERY, 1973). This procedure involves incubating a detergent extract with excess 1~IßBuTX and pipetting an 0" lml aliquot of the incubation mhcture onto DBAEcellulose disq (whatman DE 81, 2"4 car diameter) from which free toxin is easily removed by washing in 10 mM sodium phosphate, pH 7"4; 0"1 ~ Triton X-100. Receptor-bound toxin is retained and can be quantitated by counting the washed disc in a dioxane-based liquid scintillation cocktail . Both typen of binding assays wen carried out at low ionic atnngthin orderto increase therate of receptor-toxin association

a-Hungarotoxin Receptors in the Rat

24 7

which, in the case of Torpedo electric organ and rat brain, is sensitive to the concentration of rations in the medium (SCFIINmT and RAP78RY, 1974 ; McQUARRIE et a1.,1976) . Protein was determined by the method of LowRY et al. (1951) . RESULTS AND DISCUSSION The distribution of aBuTX binding sites in the central nervous system observed in the present study (Table 1) agrees with preliminary biochemical (SALVATERRA et al., 1975) and autoradiographic studies (Potz-TEIExA et al., 1975; SILVER and BILLIAR, 1976), but is TAHLE I . CONCENTRATION OP a-HUNOAROTOXIN HIImIA'ß IN VARIOUS RAT HRAIN REGIONS Area Colliculus inferior Collieulus superior Hippocampus Septum Diencephalon (with thalamus, hypothalamus) Mesencephalon (without Colliculi) Rhombencephalon (with pons and medulla) Cerebral Cortex Olfactory lobes Spinal cord (cervical and thoracic) Gasserien ganglion Cerebellum Caudate nucleus

STIPE

Concentration (fmole/mg protein) 52~3 f 0~4 (2) S1~7 f 6~6 (3) 34~9 f 3~0 (2) 27~8 (1) 25~5 ~ 2~7 (4) 24~6 ~ 1~3 (2) 22~0 ~ 3~0 (4) 18~2 ~ 4~5 (3) 14~4 f 3~1 (3) 11~7 f 1~4 (4) 7~3 f 3~5 (4) 4-2 f 0~8 (2) 1~0 f 1~4 (2)

Toxin binding activity and protein were determined as described in Materials and Methods . All values are corrected for nonspecific binding by subtraction of controls obtained in the presence of a several hundred fold excess of noniodinated toxin . Means and standard deviations are given . Number of experiments indicated in parenthesis . difficult to correlate with neurophysiological results, because little is known about specific nicotinic acetylcholine receptors in the mammalian central nervous system . Moreover, most of the work on central cholinergic synapses has been done with cat brain and spinal cord preparations, whose neuropharmacological properties maydiRer from those of corresponding regions in the rat . Relatively few physiological studies have dealt specifically with nicotinic receptors in rat brain (for review see Kxrtrsvi~, 1975 ; PHILLIS, 1976) . In spite of this some general comments may be made . The only central neurone that is generally agreed to contain a nicotinic receptor is the Renshaw cell (PfuL.1.IS, 1976). Low but significant levels were indeed found in the spinal cord in the present study. Another area of interest is the hypothalamus where acetylcholine receptors may be involved in the antidiuretic response to nicotine (Volts and KOELLE, 1970). Fairly high concentrations of toxin binding sites were observed in the diencephalonthalamus-hypothalamus section of rat brain, in confirmation of previous autoradiographic investigations (Potz-Ts1sxA et al., 1975; SILVER and BILLIAR, 1976) . Similarly, the hippocampus is assumed to contain acetylcholine receptors of the nicotinic type . Not only is the septo-hippocampal pathway one of the best-established cholinergic connections in the central nervous system (Lswrs et al., 1967), there is also evidence for the nicotinic specificity of some of the cholinoceptive sites . Thus, in his review of the pharmacology of cholinergic

248

N . SCHECHTER, I . C . HANDY, L. PEZZEMENTI and J . SCHMIDT

systems, BRIMBLECOMHE (1974) writes that `the sites of action of the drug (i.e. nicotine) are not yet entirely clear although subcortical structures, particularly the reticular formation and the hippocampus are certainly involved' . ScaMtTexLöw et al. (1967) observed that, upon i .v. application, radioactively labeled nicotine accumulates in the cellular layers of the hippocampus. This finding has meanwhile been analyzed by electron microscopic autoradiography which revealed that toxin receptors are predominantly located over synaptic complexes (HUNT and SCHMIDT, unpublished observations). In the cortex toxin binding activity is not unexpected since acetylcholine receptors that respond to nicotinic drugs have been observed there (S~roNE 1972). In the caudate nucleus on the other hand, acetylcholine receptors appear to be exclusively muscarinic (MCKENNAN and YORK, 1966), suggesting that aBuTX which does not bind in this region is indeed specific for nicotinic receptors, In vivo findings and aBuTX binding analyses are more difficult to correlate in other areas of the rat brain. Thus toxin receptor density is highest in the colliculi, and quite substantial in the septum and several other areas. Clearly additional information on cholinergic transmission in these structures would be highly desirable. Finally, the possibility has to be considered that some of the toxin receptors are located on glia cells ; binding sites for aBuTX have been described in glia cells surrounding the squid giant axon (VILLEGAS, 1975). We may conclude that although the paucity of physiological information rules out a detailed comparison, several instances of a positive correlation of nicotinic receptors and toxin binding activity can be pointed out; good examples being the caudate nucleus, devoid of both and the hippocampus, containing both. Certainly we can state that the concentration of toxin binding sites varies over two orders of magnitude between different brain regions. This argues for the specificity of toxin binding, but does not, of course, help t~ solve the problem of the physiological significance of the toxin receptors . Results of a survey of tissues outside of the central nervous system are shown in Table 2. Nicotinic acetylcholine receptors are known to occur on cells in autonomic ganglia and skeletal muscle (KoeLLE, 1970). This is where significant levels of toxin binding were also found. The neuromuscular junction is the `proper' target of aBuTX . The rat diaphragm has been a favorite preparation for the physiological and biochemical analysis of muscle receptors and is included in the present study as a control. Receptor concentrations which we found in the diaphragm are in good agreement with previously published values (BERG et al., 1972 ; ALDER et al ., 1974). Several other muscles were analyzed and found to contain comparable levels of aBuTX receptors. Considerable toxin binding activity was observed in autonomic ganglia where nicotinic receptors that differ pharmacologically from skeletal muscle acetylcholine receptors are known to exist (VOLLE arid KOELLE, 1970). For practical reasons the survey was limited to the more easily accessible sympathetic ganglia of the cervical and thoracic regions. A puzzling finding is the occurrence of significant levels oftoxin receptors in the Gasserian ganglion . Originally this ganglion was analyzed together with various brain sections, but when toxin binding activity was observed repeatedly, we decided to investigate dorsal root ganglia as well and comparable receptor concentrations were found. Since sensory ganglia lack synapses the question arises whether all neuronal acetylcholine receptors are in fact located at synapses. It is known that certain nonsynaptic structures display acetylcholine sensitivity, and therefore must bear some kind of acetylcholine receptor . For example, acetylcholine and nicotinic drugs such as nicotine and lobeline excite nerve endings of various mechanoreceptors (possibly including muscle spindles) as well as cutaneous thermal and pain receptors. in addition, carotid and aortic chemoreceptors are sensitive to acetyl-

249

a-Bungarotoxin Receptors in the Rat TABLE

2.

CoxcENTRATION of a-HUxcAROroxuv HINnINC sITFs IN VARIOUS ORGANS OF THE RAT

Tissue

Brain Spinal cord Retina. Gasserien ganglion Dorsal root ganglia Sympathetic ganglia Superior cervical l'aravertebral Nerve (sciatic) Striated muscle :$ Diaphragm Pectoralis Gastrocnemius Soleus Adrenal Heart Lung Thymus Spleen Liver Stomach Small intestine Ovary

Receptor density (fmole/mg tissue) 2~38 f 0~25 (24)" 0~59 f 0~ 13 (7) 0~12 f 0~06 (2) 0~81 ~ 0~57 ( 12) ^, 2'5l' 16~1 ~ 9~9 (7) 10~5 } 7~3 (.13)
(10) (2) (2) (8)


<0~02 <0~01 <0~01

*From Schmidt (1977) tAssuming a weight of ca 0~3 mg per ganglion ; receptor density per ganglion was 086 f a36 femtomoles (21) $Measured by DBA&ceUulose disc assay Toxin binding activity was measured as outlined in the Materials and Methods section. Individual experiments as well as controls for nonspecific binding were usually carried out in triplicate . Means and standard deviations are pre sented, with number of experiments indicated in parenthesis .

choline and nicotinic compounds . The physiological significance of acetylcholine binding

sites in sensory cells remains unclear since the acetylcholine sensitivity can be blocked by antagonists such as n-tubocurarine or hexamethonium without abolishing the sensitivity to physiological stimuli (for a detailed discussion, see PnlrrrAi., 1971) . It has also bcen found that cholinoceptive sites exist in unmyelinated peripheral fibers and that furthermore, a `striking parallelism' exists `between the actions of nicotine, acetylcholine, and other drugs on the sensory receptors and peripheral nerves, and their action on autonomic ganglia' (VOLLS, 1966) . It was therefore of interest to analyze nerve fibers for toxin binding activity. No such activity could be detected in homogenates of the sciatic nerve . This is in contrast to lobster axon preparations which have been reported to contain high levels of nicotinic receptors (MARQUIS et al., 1977) . All non-nervous tissues (apart from skeletal muscle) were free of detectable levels of toxin binding activity. Some of these tissues (e.g. heart, intestine) are known to contain intramural ganglia, however the concentration of ganglionic acetylcholine receptors in organ homogenates is expected to fall below the threshold of the assay employed. Two organs deserve to be mentioned in particular. The thymus has been known for alongtimeto contain striated `myoid' cells which, according to a recent report by KAO and DRAC1-rMAN (1977), are sensitive to acetylcholine and bind aBuTX over their entire surface . Using an immunoprecipitation technique LINnsz~ROM et al. (1976) had previously found receptor levels of 0-003 fmole/mg in rat thymus . Although we tried repeatedly to quantitate toxin

25 0

N . SCHECHTER, I . C . HANDY, L . PEZZEMENTI and J . SCHMIDT

binding sites in thymus homogenates, high background values precluded accurate measurements . The adrenal medulla is a sympathetic paraganglion receiving cholinergic input that acts on nicotinic receptors. With regard to the physiology and pharmacology of synaptic transmission the adrenal medulla resembles an autonomic ganglion, i .e . ganglion blockers suppress the responses to actylcholine as well as nerve stimulation (VoLLe, 1966). Toxin receptor levels found in the whole gland were over two orders of magnitude lower than those seen in sympathetic ganglia. However this constitutes a lower limit as no attempt was made to separate cortex and medulla. In the bovine gland such a separation is more easily accomplished, and the level of aBuTX receptors in the bovine adrenal medulla has been reported to be 0~9 fmole per mg tissue (W1LSOx and KIRSHNER, 1977) . This survey is limited to tissues and organs that can be readily dissected and prepared for in vitro analysis . Within these limits the correlation between the known distribution of nicotinic receptors and aBuTX binding activity is good . The observation of toxin receptors in sensory neurones is exceptional only in that the receptors involved are apparently extrasynaptic . Thus the results are consistent with the notion that all aBuTX receptors are acetylcholine receptors regardless of their location within either the cell or the organism . Acknowledgements-This investigation was supported by NSF grant BMS 7418607 and a grant from the Council of Tobacco Research, U .S .A., Inc. We thank NICK BRECHA for helping with the brain dissections and STEPHEN HUNT for reading the manuscript . REFERENCES ALPEA, R ., Lowv, J . and Sexessoz, J . (1974) Binding properties of acetykholine receptors extracted from normal and denervated rat diaphragm . FEBS Lett. 48, 130. BARTFAI, T ., BERG, P., Scxut zzHExo, M . and HEtttsttoNN, E . (1976) Isolation of a synaptic membrane fraction enriched in cholinetgic receptors by controlled phospholipase A, hydrolysis of synaptic membranes . Btochirn, btophys . Acts 426, 186 . Bent, D . K ., KELLY, R . B., SestaExr, P. B ., W n .~ »~anN, P . and Hiu.1., Z . W . (1972) . Binding of a-bungarotoxin to acetylcholine receptors in mammalian muscle . Pros. natn . Aced. Sct., U.S.A . 69, 147 . BRII~LECOM$E, R . W. (1974) Drug Actions on Cholincrgic Systems . Baltimore : University Park Press . Baowrt, D . A . and FUIdAGALLt, L . (1977) Dissociation of a-bungarotoxin binding aced receptor block in the rat superior cervical ganglion . Brain Res . 129, 165 . C~uNaEUx, J. P . (1975) The cholinergic receptor protein from fish electric organ . In : Handbook of PsycJwpharmacalogy, Vol . 6, p . 235, (IvEttsEN, L . L., IVEIt3EN, S . D . and SNVOESe, S . H ., Eds.). New York : Plenum Press . Ceou, T. C . and LEE, C . Y . (1969) Effect of whole and fractionated cobra venom on sympathetic ganglionic transmission . Eur .1. Pharnrac. 8, 326 . De BL~s, A, and MexLex, H . R . (1976). Studies on nicotinic acetylcholine receptors in mammalian brain -VI. Isolation of a membrane fraction enriched in receptor function for different neurotransmitters. BioeJttnt . Biophys. Res . Common. 71, 24 . DuoaeN, A. W., HALL, J . G . and LEE, C. Y . (1976) Alpha-bungarotoxin, cobra neurotoxin and excitation of Renshaw cells by acetylcholine . Brain Res . 107, 166. ETExovtd, V . A . and BENNETT, E . L . (1974) Nicotinic cholinergic receptor in brain detected by binding of a{'Ii)bungarotoxin. Bioehim . btophys. Acts 362, 346 . GxEENE, L . A . (1976) Binding of a-bungarotoxin to chick sympathetic ganglia : properties of the rooeptor and its rata of appearance during development . Brain Res . 111, 135 . GnEENE, L . A ., Svzxowsxi, A . J . VOC3EL, Z, and NnzENaEno, M . (1973) a-Bungarotoxin used as a probe for acetylcholine receptors of cultured neurones. Nature 243, 163 . HAU ., Z. W. (1972) Release of neurotransmitters and then interaction with receptors . A . Rev. Biochem . 41, 925 . Kwo, I . and Dteec~sAN, D . B . (1977) Thymic muscle cells bear acetyk holine receptors : possible relation to myasthenia gravis . Science 195, 74. Koet.zB, G . B . (1970) Neurohumoral transmission and the autonomic nervous system . In : The Pharmacological Basis of Therapeutics, p. 402, (GOODMAN, L . S. and Gtt usAN, A ., Eda .) . New York : Macmillan . KseNJEVtd, K . (1975) Acetykholine receptors in the vertebrate central nervous system . In : Handbook of Psychopharmaaology, p. 97, (IvexsEN, L. L. IvESesEN, S . D . and $NYDER, S . H., F.ds.) . New York : Plenum Press.

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25 1

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