Alpha-bungarotoxin binding and central nervous system nicotinic acetylcholine receptors

COMMENTARY ALPHA-BUNGAROTOXTN

BINDING AND CENTRAL NERVOUS SYSTEM NICOTINIC ACETYLCHOLINE RECEPTORS R. E. OSWALD’ and J. A. FREEMAN

Departments of Biochemistry, Anatomy and Ophthalmology, Vanderbilt University Medical School Nashville. Tennessee 37232, U.S.A. Specificity of alpha-bungarotoxin Ganglionic receptors Central nervous system Characteristics of alpha-bungarotoxin binding Multiple binding affinities Molecular properties of the alpha-bungarotoxin binding protein Subcellular and regional localization Developmental and experimentally induced changes in alpha-bungarotoxin Degradation rate of alpha-bungarotoxin binding sites Possible functions of the alpha-bungarotoxin binding site Conclusions

THE DISCOVERY that the nicotinic acetylcholine receptor could be selectively labeled with elapid alphaneurotoxins (LEE, 1970; CHANCEUX,KASAJ & LEE, 1970; MILEDI& PO?TER, 1971) has made possible the purification and extensive characterization of skeletal muscle (FAMBROUGH,1979) and electroplaque (HEIDMANN I% CHANGEUX. 1978) receptors (for earlier review see FEWTRELL. 1976, ed.). The polypeptide chain comprising the acetylcholine binding site has been identified (SOBEL,WEBER& CHANGEUX, 1977: DAMLE & KARLIN, 1978; and MGORE & RAFTERY, 1979) and its allosteric mechanisms (HEIDMANN& CHANGEUX,1979a,b) and interactions with other peptide chains (RAFTERY,BLANCHARD,ELLIOT,HAUTIG. MOORE,QUAST,SCHIMERLIE, WITZEMAN& Wu, 1979; OSWALD, S~BEL.WAKSMAN,ROQUES& CHANGEUX. 198Ob)are beginning to be elucidated. The binding of [‘zslJalpha-bungarotoxin to the muscle and electroplaque acetylcholine receptors is highly specific and essentially irreversible in nondenaturing conditions. By analogy, alpha-bungarotoxin has been used as a potential probe for nicotinic acetylcholine receptors in a variety of other tissues including vertebrate (SALVATERRA & MOORE, 1973;

binding

GEPNER, TENG & HALL.

1977; THOMAS, BRAI)Y & TOWNSEL,1978) brain, retina (WANG & SCHMIDT. 1976; VOGEL & NIRENBERC,1976; YAZULLA & SCHMIDT,

1976, 1977) and parasympathetic (RAVDIN & BERG, 1979) and sympathetic (PATRICK& STALLCUP, 1977a,h; CARBONETTO, FAMBROUGH & MULLER, 1978; KOUVELAS, DICHTER & GREENE, 1978) ganglia

and cultured neurons. The study of acetylcholine receptors of neuronal tissue has not progressed as rapidly as that from electroplaque due to the lower concentrations of receptor (X0- to 1000-fold less than in electroplaques) and concerns about the specificity of alpha-neurotoxins. Nevertheless, a large amount of data has been obtained on putative neuronal nicotinic acetylcholine receptors using alpha-bungarotoxin as a probe. which has revealed a number of similarities between alpha-bungarotoxin binding proteins of neuronal and muscle tissue. We review here the evidence regarding the relationship between alpha-bungarotoxin binding sites and nicotinic acetylcholine receptors in neuronal tissue and the results of studies characterizing the putative nicotinic acetylcholine receptors in the central nervous system using alphabungarotoxin as a probe. These studies have yielded a ETEROVIC & BENNETT, 1974; SALVATERRA & MAHLER, great deal of information concerning the binding par1976; MCQUARRIE,SALVATERRA, DE BLAS,ROUTES& ameters of alpha-bungarotoxin and other cholinergic MAHLER.1976; MOORE& BRADY, 1976, 1977; LOWY, agents, the possible conformational states and molMCGREGOR,ROSEMTONE & SCHMIDT, 1976; Kouecular characteristics of the protein, the receptor’s reVELAS & GREENE, 1976: OSWALD & FREEMAN, 1977: gional distribution, and developmentally and experi1979; 19804 and invertebrate (SCHMIDT-NIELSEN, mentally induced changes in the molecule. The evidence supports the conclusion that alpha-bungarotoxin does bind to nicotinic acetylcholine receptors in ’ Present address: Neurobiologie Molt5culaire. Dtpartesome parts of the central nervous system: however, its ment de Biologic MolCculaire, Institut Pasteur. 25, Rue du Dr. Roux, 75015 Paris. France ability to block synaptic transmission (particularly in NX‘.

6. I

$4

I

spinal ganglia) may vary with different species. This review willconcentrate mainly on the vertebrate brain receptor (including retinal receptors), although some aspects of gangtionic receptors will also be considered. SPECIFlCfTY

OF ALPHA-BUNGAROTOXIN

Ganglionic receptors The specificity of alpha-bungarotoxin and other snake alpha-neurotox~ns as markers for ~~~ro~a~ acety~cho~ne receptors has been questioned (Cwor; & LEE, 1969) since the introduction of the toxins as markers for muscle and electroplaque receptors. C~ou & LEE (1969) demonstrated that transmission through the sym~theti~ ganglia of the cat is not affected by the local applicatjon of cobra alpha-neuron toxin. Since that time, a number of laboratories have reported the absence of inhibjtjon of synaptic transmission by alpha-neurotoxins (e.g. alpha-bungarotoxin) in chick sympathetic neurons (CARBONETTOcr al.. 1978; KOUVELASer al., 197X)* chick ciliary ganglion neurons (RAVDIN & BERG, 1979), guinea-pig myenteric plexus (B~RSZ~~AN & CERSHOFJ.1977), rat superior cervical ganglion neurons (Ko, BURTON & BUNGE,1976; BROWN& FUMAGALLI, 1977; NURSE& O’LAGUE, 1975; OBATA, 1974) and a rat sympathetic neuron cell line (PATRICK & STALLCUP, 1977u.h). When superior cervical gangi~on neurons are cuttured with skeletal myotubes, choIinergic synapses are formed between neurons and between neurons and muscle cells (O’LAGUE OBATA, CLAUDE, F~JRSHPAN & PomR, 1974). Within the same culture, alpha-bungarotoxin abolishes transmission between nerve-muscle synapses but not nerve-nerve synapses (?&JR% & O’LAGUE, 1975). These results, the radioautographjc demonstrations of alpha”b~~ngarotoxin binding sites in gangiionic neurons (FUMAGALLI, DF REWIS & MIANI, 1976) by in viva application, and the electronmicroscopic studies (BUKZTAIAN& GERSHON, 1977) of the ~netration of ~anth~um (a cation with a Stokes radius similar to aIpba-buagarotoxin). suggest that the lack of inhibition by alpha-bungarotoxin is not due to the presence of a diffusion barrier. CHIAPPINELLI & ZICMOND(1978) have reported the inhibition of synaptic transmission by alpha-bungarotoxin in the chick ciliary ganglion. This may represent inactivation of a gang~ionic nicotin~c acetyl~ho~ine receptor by alpha-bungarotoxin, although high concentrations of toxin (1 micromoiax) were required and the results were variable among different batches of toxin. An alternative explanation is that a contaminant in the toxin prepa~tion, such as peak Hi-1 (LEE, tb~1-83, KAU 6% S~IN~~UI, 1972) of 3u~g~rus multicinctus venom which has been shown to block ganglionic synaptic transm.~ssio~ (RAVDIN & BERG. 1979). may have been responsible for the inhibition. On the other hand. CONTI-TR~XXONI, GOIX, PACCI & RO~SI (1979) have observed an inhibition of synaptic transmission in chick ciliary ganglia using a homologous

alpha-neurot~xin purified from -Cclcr nyltr sia,nc’n.sl,< venom. These experiments suggest the possibility that parasympathetic ganghonic receptors might differ from sympathcti~ gangI~oni~ receptors in their sensi.. tivity to alpha-bu~garotox~Il~ A number of studies have reported specific bmdmg sites for al~~h~~-bungaroto~in in ganghonir: neurons (GREEM. 1976; PATRKK & STALIXXV. 197711.h:li;or”VIUS P! (II.. 197X; RAVIXX & BERL;. 1979: CARRO_, NET7‘To rl 01.. 197X: FU~~AGALI.I ~7

ti/ . IWh: C~KEWE, Volt. & NIRESBERG. IW.3); however, some evidence suggests that the ~~~romolecu~e(s) to which aIpha-bungarotoxin binds may be different from the acetylcholine receptor. Wexamethonium. which is a potent antagonist of ganglionic cholmergic tr~il~smjssion, is it weak inhjbitor of alpha-bungarotoxin binding (PATRICK& STALLU.:P.197%; GREEN it cil., 1973). altltou~h this mighi tx expected were hexamethonium to exert its effect on gangli(~~~~~ neurons by selectively binding to the Ionic channels opened by acetylcholine (ionophores) rather than by direct ~ompetitio~l at the receptor ~~~~~K~~~, 1970: Asc~r:.R. LAKCI:& Rnr~;c;, 1979). On the other hand, an jrnrnunoiogj~~ study by PAmri K & Swr.t.(~l~ (19770) indicated that antibodies to eel ~~e~tropl~~qu~ acetylcholine reLvptor block agonist-induced sodium Hux m cultured ganglionic cells. but the antibody does not precipitate the alpha-bungarot~~xi~~ binding componcnt. They also demonstrated that detergent extracts of the cells could inhibit the ability of the antibody to recognize [ ‘z5f3alphii-b~in~rl)toxin--a~etylcholine receptor of muscle. These rosults could indicate that alpha-bungarotoxin binds to a molecule other than the gangli~ni~ nicotirG.~ ~~~ety~choline receptor or that the ~~~Iioni~ receptor consists of two subunits that dissociate when extracted by detcrgent, one of which contains the ~ultige~li~ determmants that are recognized by eel anti-receptor snribodies and the other of which Carrie:, the alpha-bungarotoxin binding site (PATRICK ~$2STAI,I.U:P. I977a). In contrast to the studies cited ahovc, in which alpl~~-bungarot~~xitl was found to be ineffective in blocking responses to ~~~etyl~ho~i~l~of sympat.heti~ ganglion neurons in several different spt~ies and in different culture conditions. MARSHAI.L(1979) has convincingly demonstrated the effectiveness of alphabungarotoxin (1 x IO -‘u) tn blocking ni~o?ini~ trat~sm~ssion at frog sym~athetjc ~d~~~lior~neurons. I[n the same study, both ~rox~dase-dabbled alpha-bungarotoxin and peroxidase-labeled antibodies against Torpedo acetylcholine receptor were shown to bind to the same subsynaptic JegiOnS hCah?d benmth synaptic boutons. Some possible reasons for the tissue and species differences in the ability of alpha-bung~rotoxin to block nicotinic transmi~~~o~~are discussed in the next section. SYTKOWSKJ,

Aip~~-bun~arotox~ is without o@ct on synaptic tr~~nsmjssi~)l~at nicotinic ch~~l~ner~l~synapses in the

Nicotinic acetylcholine receptors in the CNS

3

mission, preparations exhibiting a higher affinity for alpha-bungarotoxin would be expected to contain neurons whose response to acetylcholine is inhibited by alpha-bungarotoxin. To a first approximation, this supposition seems to be true. Systems such as electroplaque. skeletal muscle, and the retinotectal synapse of lower vertebrates, where alpha-bungarotoxin is effective in inhibiting synaptic transmission, contain binding components from which alpha-bungarotoxin dissociates very slowly. On the other hand, in the mammalian central nervous system, where no effect of alpha-bungarotoxin on synaptic transmission PANIAK (1975) showed that a related neurotoxin from dissociates the venom of the elapid snake Dendroaspis uiridis has been observed, alpha-bungarotoxin quite rapidly (reviewed below). (4.7.3) is capable of blocking nicotinic cholinergic Alpha-bungarotoxin blocks retinotectal synaptic transmission both in the frog spinal cord and at the frog neuromuscular junction. In a binding study with transmission in the toad (FREEMAN,1977; FREEMAN. SCHMIDT& OSWALD, 1980), goldfish (SCHMIDT& [‘251]Dendroaspis toxin (dendrotoxin, 4.7.3) to rat brain, HANLEY, BENNETT & LUKASIEWI~Z (1978) OSWALD, 1978; FREEMANet al., 1980; SCHMIDT& FREEMAN.1980). turtle (SCHMIDT& FREEMAN,1979). found two dendrotoxin binding sites for each alphaand pigeon (SCHMIDT& FREEMAN,1979). The retinobungarotoxin binding site; however, unlabeled alphatectal system of the toad and goldfish is likely to be bungarotoxin displaces all [‘251]dendrotoxin binding. cholinergic (OSWALD& FREEMAN,1977; OSWALDet These observations, in conjunction with the finding al., 1979; SCHMIDT& FREEMAN,1980) so that the that dendrotoxin and alpha-bungarotoxin binding effect of alpha-bungarotoxin seems to be due to its sites copurify, led HANDLEYet al. (1978) to suggest binding to a nicotinic acetylcholine receptor. In addithat both toxins bind to the nicotinic acetylcholine tion. alpha-bungarotoxin is effective in blocking both receptor, and that the extra sites available for dendrointracellular and extracellular responses of goldfish toxin binding may be related to the receptor’s active tectal neurons to the iontophoretic application of site. Immunological studies have provided some evi- acetylcholine (FREEMAN,1979a,h). However, SCHMIDT dence that the alpha-bungarotoxin binding site of & FREEMAN(1980) found no evidence for alpha-bungarotoxin inhibition of retinotectal synaptic transmammalian brain is carried on a molecule with antimission in the rat brain. As mentioned previously, the genie similarities to the Torpedo electroplaque acetylcholine receptor. Both P. SALVATERRA (personal com- ability of alpha-bungarotoxin to block synaptic transmission in the central nervous system may be related munication) and BLOCK& BILLIAR(1979) have reported to the multiple binding sites and slow dissociation some cross reactivity between anti-Torpedo sera and found in tissues where the inhibition is observed (see the mammalian alpha-bungarotoxin binding protein. These results, however. have been questioned by below). MORLEY& KEMP(1980) who have found no evidence for cross-reactivity between the mammalian brain CHARACTERISTICS OF ALPHA-BUNGAROTOXIN alpha-bungarotoxin binding protein and either antiBINDING EIectrophorus or anti-Torpedo electroplaque acetylSeveral early estimates of the concentration of choline receptor sera. Thus, the question of whether alpha-bungarotoxin binding sites in mammalian brain alpha-bungarotoxin binds to the CNS nicotinic were much too high (17.5 nmols/g of guinea-pig coracetylcholine receptor ha> not been fully resolved. Im1972; and 625 pmolsig of mouse brain, munological and dendrotoxin binding studies suggest tex, BOSMANN. an identity between the toxin binding site and the SCHLEIFER & ELDEFRAWI,1974). More recent results acetylcholine receptor. However, studies of the ability suggest that most vertebrate whole brain tissue conof alpha-bungarotoxin to inhibit synaptic transtains between 1 and 10pmols of alpha-bungarotoxin mission have produced conflicting results. The ability binding sites per gram of tissue (SALVATERRA& to inhibit synaptic transmission may be highly &penMOORE,1973; SALVATERRA. MAHLER& MOORE,1975: dent on the tissue and species under study. Most MOORE& BRADY,1976; LOWYet al., 1976; KOUVELAS likely during the course of vertebrate evolution. the & GREENE,1976). These values are consistent with an portion of the molecule that binds acetylcholine has estimate of 3.1 pmols of C3H]nicotine binding sites been rigidly preserved due to its importance in per gram of mouse brain (SCHLEIFER& ELDEFRAWI, physiological function, whereas the portions of the 1974) and are several orders of magnitude lower than molecule that function in the auxillary interactions the concentration of acetylcholine receptors in Tarwith alpha-bungarotoxin may be of less importance pedo electroplaque (RANG, 1975: COHEN & CHANand thus may have randomly varied. Assuming that GEUX, 1975). the strength of these auxillary interactions is related The binding of alpha-bungarotoxin to central nerto the ability of the toxin to inhibit synaptic transvous system nicotinic acetylcholine receptors displays

frog spinal cord (MILEDI & SZCZEPANIAK,1975) and in Renshaw cells of the cat spinal cord (DUGGAN, HALL & LEE, 1976~). The failure of alpha-bungarotoxin to block synaptic transmission at Renshaw cells was originally thought to be due to the failure of the toxin to bind to the cells (DUGGAN,HALL, HEADLEY, HENDRY & MINCHIN, 1976b); however, the recent evidence of HUNT & SCHMIDT(1978) suggests that alphabungarotoxin does bind to Renshaw cells in the rat. While alpha-bungarotoxin does not inhibit synaptic transmission in the frog spinal cord, MILEDI& SZCZE-

some similarities to the binding of alpha-bungarotoxin to skeletal muscle and electroplayue acctylcholine receptors, although significant differences have been observed. The apparent equilibrium dissociation constants for alpha-bungarotoxin binding to both muscle and electroplaque acetylcholine receptor (MAELICKE, FULPIUS, KLCTT & REICH,1977; RAFTEKY, SCHMIDT, CLARK & Wo~corr, 1971) and central nervous system (LUKASIEWICZ& BENNETT, 1978 : ETEROvre & BENNETT, 1974; M(X)RE & BRAD\. 1976: 1977; SALVATERRA& MAHI.ER. 1976; MCQUARRIE. or ~11.. 1976; MCGEER, MCGEER & INNANEN, 1979: and KOUVELAS & GREENE, 1976) and retinal (WANG & SCHMIDT, 1976: VO(;EL & NIRENBERG. 1976) nicotinic acetylcholine receptors are in the range of 10 ’ to IO-@ M. Exceptions include the report of a h;,, of 40 pM for the rat brain [’ 2”I]alpha-bungarotoxin binding protein (LOWY er ul.. 1976) and a report of a Kn of approximately 10OpM for the alpha-bungarotoxin binding protein from goldfish brain (OSWALD & FREEMAN, 1980~). Dissociation kinetics, on the other hand, are quite different. The binding of alpha-bungarotoxin to electroplaque acetylcholine receptor is essentially irreversible (RANG, 1975; COHEN & CHANGEUX. 1975). and the binding in skeletal muscle is only very slowly reversible (tli2 > 100 h) (BROCKES & HALL, 19750: COLQUHOUN& RANG, 1976). Alpha-bungarotoxin dissociation from mammalian and avian central nervous system and retinal nicotinic acetylcholine receptors has been reported to be much more rapid, with half times of 5-15 h (Lowu et ul., 1976; SALVATERRA& MAHLER, 1976; K~UVELAS & GREENE. 1976; VOGEL & NIRENBERG, 1976). One exception is the report of a dissociation half-time of 62.3 h for rat brain receptor (LUKASIEWICZ& BENNETT. 1978). The dissociation of alpha-bungarotoxin from toad (OSWALD, 1979) and goldfish (OSWALD & FREEMAN. 1979) brain is biphasic with slowly dissociating components having fl .* values of 30-45 h. respectively. This heterogeneity seems to be due to two interconvertible [‘251]alphabungarotoxin binding sites rather than to two distinct molecules (OSWALD & FREEMAN, 1980a). The ability of [’ 251]alpha-bungarotoxin to recognize this ‘high affinity form’ of the molecule may be related to the to inhibit synapability of [ 1251]alpha-bungarotoxin tic transmission in the toad and goldfish brain (see ‘specificity of alpha-bungarotoxin’). Pharmacological studies of brain and retinal tucotinit acetylcholine receptors (SALVATERRA& MOORE. 1973; SALVATERRAet al., 1975; VOGEL & NIRENBERG. 1976; SALVATERRA& MAHLER, 1976; SPETH, CHEN. LINDSTROM.KOBAYASHI& YAMAMURA,1977: MOORE& BRADY, 1976; 1977; MORLEY, LORDEN. BROWN. KEMP & BRADLEY, 1977; YAZULLA & SCHMIDT, 1977: SCHMIDT, 1977; KOUVELAS& GREENE, 1976, OSWALD & FREEMAN, 1979; 1980a) have all found that nicotinit (DALE. 1914) cholinergic agents inhibit the binding of alpha-bungarotoxin. Exceptions include the potent neuromuscular depolarizing blocker, decamethonium.

which has only a very weak eff’ect on itipha-bungarotoxin binding. and hexamethonium. which inhibits transmission in the goldfish brain (OSWALD & FKEL. MAN. 1979; 1980~) but not ial brain (MOOKI $2 BRAD\. 1976). Also, nicotine. a potent _3gonist, has no effect on the binding of alpha-butig~lrotoxln 1~; the outer plexiform layer of the chick r\:rina, whereac it potently inhibits binding to the tnnet pleGform iayrr (YAZULLA & SCHMIDT. 1977). Muscariruc agents. SUCII ‘ts atropine, have typically been found to be +.mt! i ttri weak inhibitors of alpha-bullgarnto~rr~ binding (iii, values of 1 ttr IOmM). As discussed above, the alplla-hun~arottxin hmdmg properties of electropiaque snif muscle ,~cct>l. choline receptors differ somewhat from central IYXVOM system alpha-bungarotoxin binding proteins. The similarities with respect to the mcotinic cholinergic nature of the binding and the ,lpparcnt equilthrium dissociation constants lend crcdencc to the argument that the central nervous system alpha-bungarotoxin binding protein is a nlcotinrc acetylcholine receptor.

Binding affinity heterogeneity hat been reported both with respect to cholinergic agonist binding (LUKAS, MORIMOTO & BENNETT, 1979: OSWALL) & FREEMAN,1980a) and [ ’ 251]alpha-bungarotoxin binding (OSWALD & FREEMAN. 1979; 198Oa). The inhibition of [3H]alpha-bungarotoxin binding has been used as a medSUre of cholinergic agonist binding to the rat brain receptor (LUKAS cr rri,. 1979: MILIEH. LUCAS & BENNEI~~,1979). These cxperlments demonstrate that the receptor exhibits a time-dependent mcrease in affinity for agonists that is thought to be related to physiologically observed receptor desensitization (HEIDMANN & CHANGEIIX. 1978). The effect requires the presence of calcium ions. and thioi groups seem to be involved. These findings are %ery significant in that they suggest that the important control mechanisms that are known to exist for muscle and electroplayue acetylcholine receptors (reviewed by HEIDMANN& CHANGEIIX. 1978) may also be present in central nervous system receptors. As noted above, the binding of alpha-bungarotoxin to both the toad (OSWALD. 1979) and goldfish (OSWALD & FREEMAN, 1979; 1980~) brain shows the presence of multiple binding affinities in both equilibrium binding and dissociation krnctics studies- The dissociation kinetics of the goldfish brain receptor were studied by the addition of a lrOOO-fold excess of equilibrated previously alpha-bungarotoxin to receptor [12sI]alpha-bungarotoxin--acetylcholinc complexes. A slowly dissociating component (II : of 44 h) constituting 80% of the binding sites and a rapid dissociation component (t,,2 of I7 min) constituting 20% of the sites were found (OSWALD & FREEMAN. 1979). When either carbamylcholine or d-tubocurarme are added instead of cold alpha-hungarotoxin. the percentage of rapidly dissociating sites increase5 at

Nicotiilic ac~tylcholine receptors in the CNS

the expense of slowly dissociating sites with very little change in the kinetic rate constants (OSWALD& FREEMAN, 19800). The increase in the number of low affinity sites is a saturable function of both carbamylcholine and d-tubocurarine concentration, having KD’S several orders of magnitude higher than the Kg for inhibition of [‘251]alpha-bungarotoxin binding. This suggests that the effects are mediated through an allosteric binding site. These results were interpreted as indicating the presence of two interconvertible aflinities for alpha-bLingarotoxin.

MOLECULAR PROPERTIES OF THE ALPHABUNGAROTOXIN

BINDING

PROTEIN

Studies of the molecular properties of central nervous system alpha-bungarotoxin binding proteins have been limited due to the small quantities of highly purified receptor available. Experiments have been performed using radioactively labeled alpha-bungarotoxin as a marker for the receptor in velocity sedimentation analyses in sucrose gradients (SALVATERRA & MAHLER. 1976; Lowv &

ri al., 1976; SETO, ARIMATSU

AMAND,1977; OSWALD& FREEMAN,1979), gel filtration (SALVATERRA & MAHLER, 1976; Lowy et ul., 1976; SETCIet ul., 1977; MOORE & Lou, 1972; OSWALD & FREEMAN,3979), diffusion analysis (OSWALD & FREEMAN,19793, isoelectric focusing (LOWY et al., 1976; SETO et al.. 1977; BOSMANN,1972; OSWALD& FREEMAN,1979). and affinity chromatography on lectin--sepharose columns (SALVATERRA,GURD & MAHLER,1977; OSWALD& FREEMAN,1980~). The velocity sedimentation coeflicient of the alphabungarotoxin-receptor complex of mouse and rat brain has been estimated to be 11.4 S (LOWY et ul., 1976: SETO et d., 1977) and 12.9 S (SALVATERRA & MAHLER, 1976). SALVATERRA& MAHLER (1976), employing sucrose-D,0 gradients, calculated a partial specific volume of 0.8 cm3/g, indicating a considerable amount of detergent binding. When the protein partial specific volume of 0.72.5cm3,/g was assumed and the data combined with gel filtration analysis (see below), a molecular weight of 357,OOQ was calculated. Similar results were obtained by OSWALD & FK~I-:MAN(1979) for the nicotinic acetylcholine receptor solubilized by Triton X- 100 from the goldfish brain. using the technique of SMMIGEL & FLEJSCHEK(1977) to estimate the partial specific volume and sedimentation coefficient. The toxinreceptor-detergent complex has a partial specific volume of 0.79 cm2/g, and an sZO,wof 11.45 S. The molecular weight of the toxin--receptor complex (assuming a protein partial specific volume of 0.735 and using estimates of the Stokes radius from gel filtration and diffusion analysis) is 340,000. Early studies of the central nervous system alphabungarotoxin binding protein indicated that it behaved as a globular protein with a molecular weight in the range of 50,000 to 80,ooO (hog brain;

5

MWRE & Lou, 1972). More recent evidence, however, suggests that the receptor from mouse, rat and goldfish brain co-chromatographs with Torpedo electroplax acetylcholine receptor, behaving as a globular protein with a molecular weight of approximately 500,000 and a Stokes radius of 7 to 8 nm (LOWY et al., 1976; SALVATERRA & MAHLER.1976; and SETOet al., 1977; OSWALD & FREEMAN,1979). OSWALD& FRIEEMAN(1979) have explored the possibility that gel filtration underestimated the Stokes radius (NOZAKI, SCHECHTER. REYNOLDS & TANFORI),1976) because of anomalous behavior arising from apparent asymmetry of the molecule (frictional coefficients f 2.5 for rat brain, SALVATERRA & MAHLER, 1976; 1.3 to 1.5 for goldfish brain, OSWALD& FREEMAN,1979). Free diffusion analysis (OSWALD& FREEMAN,1979) yielded a D 2o.w for the toxin-r~eptor-detergent complex of 2.85 + 0.54 x lo-’ cm2/s which corresponds to a Stokes radius of 7.4 f 0.4 nm, in close agreement with the value obtained from gel filtration. Isoelectric focusing has shown that the central nervous system alpha-bungarotoxin binding protein has an isoelectric point of approximately 5 (BOSMANN, 1972; LOWY et al, 1976; SETO et al., 1977; OSWALD & FREEMAN,1979), as does the acetylcholine receptor from muscle (BROCKES& HALL, 197%) and electroplaque (RAFTERYet al.. 1971; RAFTERY,SCHMIDT, MARTINEZ-CARRION, MOODY, VANDLEN & DLJGUID, 1973; BIESECKER. 1973; TEICHBERG & CHANGEUX, 1976). Estimates include 4.8 (guinea-pig cerebral cortex ; BOSMANN,1972) and 5.6 (mouse brain ; SETO rt & 1977), and 5.0 (goldfish brain; OSWALD& FREEMAN, 1979) for the toxin-receptor complex, and 4.9 (rat brain; LOWYet al.. 1976) for the receptor itself, Isoelectric points of the toxin-receptor complex are slightly higher than the receptor itself due to the highly basic nature of the alpha-bungarotoxin molecule (Tu, 1973). The alpha-bungarotoxin binding protein from rat brain is retained on Concanvalin A- and wheat germ agglutinjn-Sepharose columns and to a lesser extent on ~ici~~~ ~u~~t~~~.~lectin-Sepharose (SALVATERRA et ~11,1977). No interaction was detected with a fucose binding protein-Sepharose column. This suggests that the receptor is a glycoprotein with mannose, ,Y-acetyl glucosamine and galactose. but not fucose residues. The interaction with Conanavalin A has been confirmed by SETOec ~1.i1977), MO(~RE& BRADY(19773, and OSWALD& FREEMAN(1980a). Some evidence of microheterogeneous glycosylation exists (OSWALD & FREEMAN, 1980a) for the goldfish brain nicotinic acetylcholine receptor. These findings are consistent with the results of direct carbohydrate analysis of a~tylcholine receptors from Torpedo ~~~~~~~~~e~ electroplaque (RAFTERY, VANDLEN, REED & LEE, f 976). The limited information on the molecular properties of the central nervous system alpha-bungarotoxin binding protein suggests that it is an acidic glycoprotein with a molecular weight of approximately 350,ooO. It binds a considerable amount of detergent

6

R. t

OSWALI)

and J. 4.

(weight fraction of 0.35, SALVATERRA & MAHLER, 1976; 0.30, OSWALD & FREEMAN. 1979) and is very asymmetric. A more detailed analysis will require more efficient purification procedures, a richer source of central nervous system receptor or more sensitive analysis techniques.

F'KWMAX

spatial resolution but is less quantitative than dissection and binding assays. HUNT & SCHMIDT (1979) have made a comprehensive radioaurographic analysis of alpha-bungarotoxin binding patterns in the rat central nervous system. The major conclusions were that the greatest amount of binding OL’CUIX in sensor> areas (e.g. superior colliculus [the homologue of the optic tectum in lower vertebrates]. olfactory bulbs. ventral lateral geniculate nucleus, sensory nucleus oi SUBCELLULAR AND REGIONAL LOCALIZATION the trigeminal nerve) and limbic areah (hlppocampus. Studies of the subcellular distribution of the alphaamygdala. olfactory tubercle, medial mammillar~ nubungarotoxin binding protein of rat brain (SALVA- cleus. and the dorsal tegmental nuclt;:is g,f Gudden). TERRA et al., 1975 ; ETEROVIC & BENNETT, 1974; TIT+ In addition, the dorsal motor nucic:t> c-,i the lagus DALL,KENT, BA~KIN & ROSENBERG,1978) have indinerve, the nucleus ambiguus. and the interior and accated that the toxin binds mainly to the synaptosomal cessory olivary nuclei were labeled. SIA.~EK& BILLIAR fraction (GRAY & WHITTAKER,1962). This finding is (1976) also found label in the hippocampus and amygconsistent with the binding of C3H]nicotine to the dala: and POLZ-TUERA, SCH~L)T & KAKTI:V (1975) synaptosomal fraction of rat brain (YOSHIDA & IM- reported labei in the superior colliculus, the ventral URA. 1979), and with the electron microscopic localizlateral geniculate nucleus. the hippocampus and the ation of horseradish peroxidase labeled alpha-bungarodorsal motor nucleus of the vagus ncr:v. toxin binding at synaptic junctions in rat brain The radioautographic localization %rfjiL’I]alpha(LENTZ & CHESTER,1977) and toad brain (LUTIN, bungarotoxin binding has been studied In the optic BRADY, JENSEN, SKENE & FREEMAN,1975) and of tectum of the chick (POLL-TUIIKAc’( Iti., 1975), toad [1251]alpha-bungarotoxin binding in synaptic regions (FRL:EMAN & Lk ~TIY. I Y75). goldfish (C)SWAU I, of rat (HUNT & SCHMIDT, 1978) and mouse (ARI- SCHMIDT, NORIXS & FREEMAN.IYXULIJ. and larval salamander (D. WUNK & J. A. FRE~MAX.unpublished MATSU,SETO & AMAND, 1978) brain. In addition, a significant amount of binding has been found in observations). In each case the binding pattern shows microsomal fractions (SALVATERRA et al., 1975; ET~R- discrete lamination within the tectai neuropil, with ovrc & BENNETI. 1974; DE BLAS& MAHLER, 1978). the receptors very likely being localucd on discrete DE BLAS & MAHLER (1978) isolated a microsomal regions of radial dendrites of tectal cells in the deeper cell layers (FREEMAN. 1979a.h: 1980tr.i~: PWZ-TFJERA subfraction by isopycnic sucrose gradient sedimenta~‘t cl/., 1975). The binding is localized 111iaycrs identtion that was highly enriched in receptor binding. The ified by the electrophysiological technique of current morphology, enzymatic markers, and protein content source density analysis (FREEMAN~& NICHOLSON. of this fraction indicate that it is derived from Gray’s 1975) to receive optic input. Type II synapses (GRAY, 1959). Thus, the binding of alpha-bungarotoxin in the central nervous system More detailed physiological studies ot the ceilular distribution of acctylcholine receptors have been perseems to be largely associated with synapses. formed in slices of goldfish optic tectum (FREEhfAh, The regional distribution of alpha-bungarotoxin 1979a.h: 1980a.h): the dendritic surfaces of many tecbinding sites has been studied using dissection foltal neurons, identified under Nomarskl optics. contain lowed by biochemical assay and by histological techthree discrete zones having high sensltibity to iontoniques. The results of these studies are difficult to phoretically applied acetylcholine. as shown b> compare because of the differences in dissection techintracellular recording. The same I~~~IC‘S also bind nique. In general the evidence suggests that in rat fluorochrome-conjugated alpha-hungarotoxrn. which brain, the highest levels of binding are associated with appears in discrete aggregates. the hippocampus and colliculi (SCHECTER,HANDY. Radioautographic localization of [’ “IJalpha-bunPEZZEMANTI & SCHMIDT, 1978; MORLEY et cd., 1977; garotoxin in the retina of chick (VOGLL& NIRENRIJKC;. SEGEL,DUDAI & AMSTERDAM, 1978; SAL.VATERRA & 1976: YA~LXLA & SCHMIDT. 1977) atld goldfish :ind FODERS,1979) and moderate to low levels with the cerebral cortex and cerebellum (TINDALL(‘I ul., 1978 : turtle (YAZI 1.1.~ 81 SCHMIDT.1976) has revealed that SALVATERRA etok,1975; SCHECTER et al., 1978; SEGEI. binding occurs in both the outer and Inner plexiform layers. Within the inner plexiform layer of the chick et ul., 1978). Similar results were found for mouse retina four discrete horizontal bands seem to he brain (SALVATERRA & FODERS,1979); however, in the present (VWXL & N~RENBERG, 19761.The binding in rabbit, the cerebral cortex seems to bind more toxin the outer plexiform layer is more diffuse (V
Nicatinic acetylcholine receptors in the CNS toxin binding occurs in discrete regions of the vertebrate central nervous system and that, in most cases, this binding is associated both in subcellular fractionation (SALVATERRA et al.,197.5)and regional distribution (HUNT & SCHMIDT, 1978) with synaptic structures and other cholinergic markers. In most cases, the binding seems to be associated with sensory or limbic areas.

7

trast to this report, WANG 8~ SCHMIDT(1976) found a similar pattern of increase in the total number of receptors, but observed a sharp decrease in the number immediately following hatching. The data for the optic lobes are particuIarly interesting because at 12 days iif ovo retinotectai synapses begin to appear (CANIWO & DAMO,

1973; CRO.SSLANI).COWAN &

ROGERS,1975). Since [12~I]alpha-bungarotoxin binding has been localized to layers receiving retinal terminals (PoLz-TEJERAet al., 1975: HUNT & WEBSTER, 1975) and some evidence is available suggesting that DEVELOPMENTAL AND EXPERIMENTALLY ~~iph~bungarotoxin may be localized at retinotectal INDUCED CHANGES IN ALPHAsynapses (BRECHA,FRANCIS& SCHECHTER, 1979), the BUNCAROTOXIN BINDING time course of formation of retinotectal synapses may be correlated with rhe appearance of alpha-bungaroThe development of vertebrate central nervous system acetylcholine receptors has been studied in the toxin binding sites. A similar situation is present in the chick retina, where receptors are synthesized prior rat and chick brains. The number of alpha-bungaroto the appearance of synapses (VOGEL& NIRE~5ERG. toxin binding sites increases gradually following birth until reaching an adult level at 10 days (SALVATERRA 1976). The observations that alpha-bungarotoxin binding sites appear at the same time as synapses in & MCXIRE, 1973). A slight decrease in the total number of sites has been observed between 37 days the chick, but appear earlier than synapses in the rat, may reflect real species differences in the maturatjon (postnatal age) and adulthood for whole rat brain (WADE & TIM~RA~,1980). Although the rather large of synaptic connections. However, alternate explanations are possibIe: the synapses to which alpha-bunbrain areas under investigation make the interpretation of these results difficult, the facts that the final garotoxin sites are localized in the rat brain may be level of acetylcholinesterase (ARDEL-LATIF,SMITH&? among the earliest to form, or alpha-bungarotoxin may be binding to different molecules in the two ELLINGTON. 1970) and number of ethanolic phosphotungstic acid staining synapses (ACHAJANIAN& tissues. Answers to these questions will require elecBLOOM,1967) is reached IS-25 days after birth suggest tron-microscopic radioautography and possibly immunoIogica1 studies to compare the two alpha-bunthe possibility that the appearance of alpha-bungarotoxin binding sites precedes synapse formation (SAL- garotoxjn birding proteins. At the neuromusc~jlar junction, den~rvation causes VATERRA & MOORE,1973). The results of the analysis a proliferation of extrajunctional receptors to occur of alpha~bungarotoxin binding to regions of rat brain suggest that the development of binding sites pro- across the surface of the muscle fiber (reviewed by gresses with a caudai to rostra1 pattern but that all FAMER~~GH.1979). The situation may be different in regions reach their adult levels by IO days following the superior cervical ganglion of the rat where prebirth (MORLEY & KEMP. 1978). The results of a ganglionic denervation is without effect on the bindradioautographic study of the development of toxin- number or distribution of alpha-bungarotoxin binding sites in the rat hippocampus (HUNT & ing sites (FUMAGALLIet al., 1976); however, the yuesbinds to the SCWMILYI’, 1979) show that the sites appear before the tion of whether alpha-bungarotoxin ingrowth of cholinergic fibers and that they develop acetylcholine receptor in ganglionic neurons confounds the interpretation of this experiment. Studies normally in the apparent absence of a cholinergic projection due to a septal lesion. Thus, it is possible of the effects of denervation on alpha-blln~drotoxjn binding sites in the central nervous system require a that receptors appear prior to the formation of synaptic connections, as shown with embryonic muscle known monosynaptic pathway with ~l~pha-bungaro~ fibers (DIAMOND& MILEDI,1962). This interpretation toxin binding sites located on the pos~synaptic memis supported by the observation that I day after birth brane and a discrete fiber bundle. such as an afferent cranial nerve, to lesion. Lesions of the fornix (DUDAI few synaptic contacts are seen in areas containing high concentrations of alpha-bungarotoxin binding & SEGAL. 1975) and septum (HUNT & SCHMIIX. 1979) sites (S. HUNT. unpublished observation in HUNT & made with the intention of denervating chofinergic SCHMII)T.1979). synapses in the hippocampus. have abolished acetylThe development of alpha-bungarotoxin binding chofinesterase activity in the hippocampus, but have sites in discrete regions of the chick brain has been no effect on the number or distribution of alphastudied by KOUVELAS& GREENE(1976). Increases in bungaroto~in binding sites. The authors (DUDAI & total receptor content occurred in ot:o between days &GAL. 1978: and HI.I~+T & SCXM~V~,1979) interpreted 12 and 19 in the brain stem and cerebellum. days I 5 these experiments as indicating that the loss of the and 19 in cerebral hemispheres and between day 12 presynaptic input does not affect the postsynaptic and hatching in the optic lobes. With the exception of receptor labeled by alpha-bungarotoxin. This concluthe brain stem. receptor content in all regions con- sion is warranted only if the alpha-bungarotoxin tinued to increase slowly following hatching, In con- binding sites are located on the postsynaptic mem-

x

R. E. OSWALD and .I. A. FREEMAN

brane apposing

a monosynaptic

tum to the hippocampus. suggest whether

input

from

No evidence

to

is present in the

such an arrangement

mammalian brain. system for the study of the effects

of alpha-bungarotoxin

appears to be the retinotectal brdtes. Electrophysiological

binding

pathway

sites

of lower

vcrtc-

studies in the toad (FRI(t,-

MAS. 1977; FREEMAN of trl., 1980) and goldfish MAS u/ ul.. 1980: SU~M~~T & indicated aptic

(FRL:L.-

FREEMAN. 1980) have

that alpha-bungarotoxin

receptor

The

binds to a postsyn-

on a monosynaptic

from the

pathway

RATE OF ALPHA-

BUNCiAROTOXIN

BINDING

degradation

of

receptors

A more favorable of denervation

DEGRADATION

the sep-

is available

rates

SITES

muscle

have been thoroughly

acetylcholine

studied

(reviewed

by

FAMBROI:G~L lY7Y. and EDWARDS. 1979). The dcgradation

rate

usually

measured

of the muscle from

acetylcholinc

[I Z’I]alpha-bungarotoxin MCMILLAN. MARSHAU SCHI~I~Z.I.

HAII..

FRAIL

DRA(.HMAN.

O’BRIEN.

lY76: &

is

bound

the muscle (PATKICK.

from

WOI.FSON & &

receptor

the loss of irreversibly

GANG

1976: &

iO7X:

FISCHBACI~.

1977: BERG & HALL.

HOGAN.

HLANG.

1975:

KAO

&

1974. 1975: Arqq.~.

retina to the tectum. In addition. the pathway can be

ASWYI.

selectively lesioned by eye removal

B~VAN. KIILHERG. LINDSTROM &

Rrc~. 1977:

DEV-

KI:OTI:S &

LIYIX:~

FAM-

crush. Eye removal toxin

binding

or

optic

ncrvc

results in a loss of alpha-bungaro-

sites in the optic

(304~~ loss; BKECHA cr ul..

tectum

1979) and

of the chick

goldfish

(40 to

M(.AI)AMs

HKOIIGH. lY7Y); b\

NIGA.

SALPU~K.

1979;

OSWALD

L’/ td..

the loss of sites closely synapses

corresponds

in the goldfish

removal

optic

with

tectum

(M~:RRA~. 1976). Conversely.

appear during (S(XE(.HTER lationship tissue

1980~). The time course of

ct crl.. 1979). The receptor

slices of goldfish

detailed tectum

chrome-conjugated

alpha-bungarotoxin.

genesis is presently

being studied.

suggest that

regenerating

tcring and the formation of acetylcholine

cells by ingrowing cord reported The

receptors

clusaggre-

on cultured

axons from embryonic discrepancy

of

muscle

chick

between

the rat hippocampus

and the chick

spinal

pathwa)

ma!

remain

shown in Xc~r~op~rskwi.s

if the

and its associated

of

of

FAMBROUGH. CHAMXUX

1.11:. C‘tIAUGEI:x

GROS.

amino

&

degradation

tern. Junction4

have 01

retinal

postsynaptic

LINIIEU

FAMHROUG~I. 1979). The

&

Junctional half-life

receptors

slight11

1980). These results

suggest that

&

the pool-

and receptor is removed with

alpha-hungarotoxin

binding

;thl> underestimate toxin dissociation

of junctional

the degradation

the

NAIII.F.R. 1978). suggesting mmal is removed. true.

these results

the control bous system Jcncrvation.

that the presynaptic

but that the postsynaptic

and the postsynaptic

receptor suggest

of acetylcholine neurons

and

arc often

(COTMAN

remain

intact.

a signiticant

In contrast to the numerous studies 01 the degradalittle

IS

known about the degradation r;tIc of neuronal

acetylcholinc receptors. CARBONI~TTO & (1970)

hvc studied the control hinding

protein

of chick

receptors

sympathetic

of stable Isotopes.

skeletal

acctylcholine

ter-

degradation

If this is

tional

receptor.

receptor.

In any

01 the ganglionic

event.

the

to the values obtained

with

measured

1I h is quite

muscle extrajunc-

receptors.

acetylcholine

rate of the gold&h

receptor

AKIAS.

~)YI.E

&

bram nicotmlc

has been estimated

& FRVI.MA~ (198Oh) with of

so that the data obtained

half time of approximatel!

The degradation

in

muscle following

As dis-

toxin binding protein of ganglionic neurons is a nico-

&

ner-

neurons

some question exists as to whether the alpha-bungaro-

similar

by central

FAMBRO~G~I

of the iilpha-bungdro-

cussed ahovc (see ‘Specificity of alpha-bungarotoxin’).

densities

difi’ercncc

& FAMreceptors

tion rate of muscle receptors cited in p;jrt above. very

may or ma! not reflect the behavior

dcaffercntation

FAMprob-

receptor\ occurs with 3

tinic acctylcholine

following

&

receptors

1979). In any event. extrajunctional

n~ot:C;ii.

receptor

due to nlpha-bungaro-

the half-life

postsynaptic

observed

studies of extra-

from the receptor (LILIIXS

other

densities

hand. have

(GARII~~.R

In the rat hippocampus. on the

vacated

the sjs-

of th< receptor b\

presynaptic terminal. hand.

is on

overestImate

due to the stabilization

using the incorporation

density

receptors

I.1 days (Cttmc; & HKANG. lY75: BEKG ni HALL lY75;

toxin

(%ORI)IZ

Of

half time of

receptors. on the other

elTczt occurs in the

~‘RI.I:MAS.

ISOtOpes

been estimated to have half-lives on the order of 5

by glia. Prelimmarq

bynaptic

lY75: MER-

19731

1976:

of txtrajunctional

and removed

cyc removal

&OS.

order of S 7 h depending on the experimental

that :I similar

tectum following

&

also been used. The

acids have

are engulfed

poldlish

stable

1975 and radioac-

rive (McHI.~~. SOBEI..

results have shown

density

to

block

WCODWAKD &

10751. &

FRYI--

the removal

membrane

receptor

half time of several days to several wcehs.

postsynaptlc

that following

of sensitivity

following (FERTMX,

alpha-bungarotoxin

&

seem to have half-lives on the order of hours. whereas

postulating intact

!GHU)I-S (1079)

presynaptlc

cell terminals

reti-

NORI)I:S. ~STHERG &

~STRERG &

the

by

observed

intact.

on 01

and goldfish

sites remain

electron-microscopically MAS (197X) and

the effects

be rcconcilcd

the return

IIKOI (;}I. 1979) and those of junctional

sites of denervation

that the alpha-bungarotoxin

ganglion

receptor

to the induction

binding

an eke both

results

by FRAWC 6t FWIIHACII (1979).

apparent

densities

and synapto-

Preliminary

alpha-bungarotoxin notcctal

In

tluoro-

of stable. high-density similar

rc-

seen

using

fibers induce

rates (FK~EMAS. lYX0~.hl clusters

temporal

aggregation.

optic

however.

H~:V+MASU.

eye

sites rc-

of the goldfish optic nerve

regeneration

between

following

lY?7:

lY76h:

iontophoresis

the loss ol

binding

ELIAS.

FAMHROLGH.

acetylcholine

60”:, loss; SCHECHTIZR. FRANCIS. DI+LI~SCI~ & GAZXA-

&

the double-label

by 0ZjWAI.I) procedure

SCHIMKE (1969) using [“HI-

and

Nicotinic acetylcholine receptors

in the CNS

9

polyionic (PFENNINGER & REES, 1976) or peptide (COTMAN & LYNCH, 1976) bonds. If this is true one would expect a high density of receptors at the subsynaptic membrane. Using physiological and anatomical information obtained in slices of goldfish optic tectum, a density of 150&3500 acetylcholine receptors/pm2 synaptic membrane has been calculated (FREEMAN, 19806). Assuming the radius of the membrane-bound receptor to be near that of the Stokes radius of the solubilized receptor (8 nm. OSWALD & FREEMAN,1979), these data suggest that acetylcholine receptors are packed very tightly at goldfish retinotectal synapses, probably constituting the major protein component of the subsynaptic membrane. Comparable data are not yet available for other CNS synapses. The release of dopamine from synaptosomes derived from the corpus striatum can be stimulated by POSSIBLE FUNCTIONS OF THE acetylcholine and this stimulation can be blocked by ALPHA-BUNGAROTOXIN BINDING SITE alpha-bungarotoxin (DE BELLEROCHE & BRADFORD, 1978). This effect was postulated to be mediated by As discussed above (see ‘Specificity of alpha-bungarotoxin’), the alpha-bungarotoxin binding protein is presynaptic nicotinic cholinergic receptors located on dopaminergic terminals. This explanation is supvery likely to be associated with the control of ionported by the observation that the selective destructranslocation at nicotinic cholinergic synaptic junction of dopaminergic terminals by 6-hydroxydotions Other functions, such as roles in the maintenance of synaptic contacts (FREEMAN, 1977; SCHMIDT, pamine reduces the number of alpha-bungarotoxin 1979) and in the presynaptic release of dopamine (DE binding sites in the corpus striatum by 50% (DE BELBELLER~CH& BRADFORD. 1978; DE BELLEROCHE, LEROCHEet al., 1979). A likely explanation for these effects is that the nicotinic cholinergic receptor is part LUGMANI& BRADFORD, 1979) have been suggested. of an axo-axonal or reciprocal synapse located on the FREEMAN(1977) found that the topical application presynaptic terminal. However, electron-microscopic of alpha-bungarotoxin to discrete regions of the toad studies (HASSLER, CHUNG. RINNE & WAGNER. 1978) optic tectum during the reinnervation of the tectum synapses after optic nerve crush prevents the maintenance of have not given any evidence for axe-axonal in the corpus striatum, suggesting the possibility that functional synaptic connections within the toxin the cholinergic receptor may have an extrasynaptic treated zone. The presynaptic terminals originally function of controlling dopamine release. invade the toxin treated zone but rather than making enduring synapses, apparently sprout terminals which CONCLUSIONS make connections on the periphery of the toxin zone. Application of alpha-bungarotoxin to normal toad The alpha-bungarotoxin binding sites in the central (FREEMAN, 1977) and goldfish (SCHMIDT, 1979) optic nervous system seem to be located on nicotinic acetyltectum evidently induces a sprouting of retinal tercholine receptors, although alpha-bungarotoxin does minals to the periphery of the zone. This effect is not block synaptic transmission at all putative central reversible within 2 weeks and is not due to cell death nervous system nicotinic cholinergic synapses, and its because tectal cells can be driven by non-cholinergic physiological effect has not been extensively tested in thalamic inputs following alpha-bungarotoxin applibrain except in the optic tecta of a few species. The cation (FREEMAN,1977). The most likely explanation binding of alpha-bungarotoxin to both particulate for these effects is that the postsynaptic receptorand detergent solubilized central nervous system alpha-bungarotoxin binding site is involved in the tissue is quite similar to its binding to electroplaque maintenance, but not the initial recognition or formaacetylcholine receptors, and the molecular properties tion, of synaptic contacts. Either the blockage of the (e.g. isoelectric point, molecular weight, and Stokes physiological activity of the receptor or the introducradius) of the central nervous system alpha-bungarotion of a high density of (positively charged) alphatoxin binding protein are similar to those of the bungarotoxin molecules to the densely packed acetylmuscle and electroplaque acetylcholine receptor. In choline receptor lattice might cause the synapse to be addition to its possible role in ion translocation, the destabilized (CHANGEUX & DANCHIN, 1976) and the acetylcholine receptor may also play a role in the axons to sprout new presynaptic terminals into toxinstabilization of synaptic connections. free regions. The latter possibility would imply that the receptor might have a structural role, possibly Ack,~nwled~ernrr2ts~This work was supported by NIH helping to maintain the cohesiveness of the presynCareer Development Award EY70240 and National Eye aptic membrane to the subsynaptic membrane via Institute Grant EYOl I 17 to JAF. [35S]methionine. OSWALD & FREEMAN (1980h) considered the potential problem of isotope reutilization in calculating the half time of degradation, which was found to be between 5 and I days assuming 20:; reutilization. These values correspond well to measurements of junctional muscle acetylcholine receptors. The difference between the degradation rates of ganglionic and brain acetylcholine receptors may reflect different functions for the two molecules. The goldfish brain receptor, like the junctional muscle receptor, seems to be involved in synaptic ion translocation, Presumably its incorporation into the postsynaptic membrane decreases its degradation rate. The rapid turnover of the ganglionic alpha-bungarotoxin binding protein may reflect an extrasynaptic function. Alternatively, the rapid turnover rate may merely be an artifact of the culturing conditions.

10

K. t; OSWAI.I) and J A. FK~I:M.~\ REFERtNCES

AB~x.L-LATIF

A. A., SMIT~I J. P. &

triphosphatase, AGHAJANIAN electron

acetylcholine

ELLING~ON

P (1970)

study.

Rrs. 6, 7 I6

Bruin

APPEL S. H.. AFJWYL R., MCAI~AMS rat myotubes

with

electron

A. & AMAND T. (1978)

microscopic

ASCHER P.. LARU

autoradiography.

ganglion

BFRG D. K. & HAI.L muscle.

Scirrlce.

BERG D. K. & HAIL rat diaphragm

synapse.

the Torpedo

itI riro

and in organ properties

Proc,. L’tlir.

culture.

nicotinic

pig cerebral

deafferented

avian

BROCKES J. P. & Purification

cortex.

of acetylcholine

receptor

from

and degradation

of protemc.

.>f the endoplaqmlc

bmdlng

SIWS 111mow<

hraln

by light

and

on the mechanism

of actmn

of acetylcholmc

antagonists

on rat

170.

I39

bound

to acetylchotine

receptors

of normal

and denervaced

from junctional

and extrajunctional

,rc:c.t~lLhollne

receptors

In

J. Ph~%r,l.. Loud. 252. 771 ~789. receptor

purified

from

.E/c(~r,op/~or~c\ <,;,s(rr~ I,.# HI,U II~,uII,s~~,I

Comparison

optic

HALL

ganglion.

I

receptor

Z. W. (197%)

HALL

receptor.

with

Rapid

Acetylcholine

in chick

CHANGNX

venoms.

networks.

between

receptor

receptor

blndlnp

actlvlt}

In the

rat

diaphragm

I

muscle

and

denervated

rat diaphragm

muscle

II

and

receptor

htock

In the rat superior

C. (1975)

nicotimc

insertion

into

receptor5

ganglia

and skeletal

chick

optic

the plasma

tectum.

E~pcrwnrtu

membrane.

29. X5 81

and turnover

of I-bungaro-

Biol. 81, 555-569

K. J. (1978) Nonequivalence Proc. rwttl.

Turnover

111Lrrtsbratc

269, 17~ 3 I.

of I-hungarotoxln

~sceptors

and acetyl-

receptors

of the r.it

Acad. Sci. U.S.A. 75, lOlO- 1020.

of junctional

and

ewtrajunctional

acetytcholinc

253, 643 644. A. (1976) Selecttve

~Vuture. Land.

stabitisation

of developing

synapses

as a mechanism

for the speciticatlon

264. 705--717 toxin

to characterlse

the chohnerglc

receptor

protem.

1247.

V. A. & ZIGMOUO R. E. (1978) z-Bungarotoxin

Proc. twtrl. Acud. Sci. L’.S.A. 75. 2999 3003. CHO~I T. C. & LOP C‘ Y. (1969) EtTect of whole Eur. J. Pharmac.

ot ,t chollnerglc

denervatrd

binding

in the developing

J. Cr/l

neurons.

Actrd. Sci. L’.S.A. 67. I24

CHIAPPINLLI

and

14, 2 t 00-2106.

Land.

CHANGE~IX J. P.. KASAI M. & Lkr: C. Y. (1970) tJse of :I snake venom Proc. mtn.

characterlstlcs

and

in normal

Biochemistr~l

J. Physiol.,

Junctions

neurons.

sympathetic

M.

Lotld.

J. P. & DANUIIN

of neuronal

receptor

14, 209-2099.

of x-bungarotoxin

Discrimmation

and cobra

sympathetic

m chick H11.41~

alpha-bunparoroxin

cholinerglc

in normal

receptors

CAKRONF~TO S. T.. FAMMROI C;H D. M. & M[XLI,K

.Vutwr.

and biochemical

loss of nicotine

receptors.

L. (1977) Dissociation

CANTINO D. & DANE~ L. S. (1973) Synaptic

C. C. &

the hypothalamic

Biochernisfry

CAKHONETT~ S. & FAMBROUGU D. M. (I 979) Synthesis.

diaphragm.

.it ‘4 h>mpathcrIc

R~1.s 129, 165~ 16X.

by alpha-bungarotoxin

receptors

of he~~mcthon~urn

Res. 178, 381~ 387.

receptors

Acetylcholine

and extrajunctional

BURSZTAJAN S. & GERSHON D. D. (1977)

receptors

action

167, 27.7 280.

[“‘I-r-bungarotouin.

Z. W. (1975h)

Brain

between

Brain

Identification

SCHE(.HTER N. (1979)

of junctional

of the blocking

simitarities

cholinergic

lobe. Bruit? Rex

BROWN D. A. & FUMAGALU cervical

potential

Sch. 48. 4~ 5

J. hiok Chem. 247. 130 145.

and interaction

BKOCKES J. P. &

Med.

R. B. (1979) Immunologic

c~ulifornicu

BRE~HA N., FRANCIS A. &

CtlANc;

A quantltatl\c

mutts. Acud. Sci. U.S.A. 74, ?13{&~2134.

of a-bungarotoxm

of the cholinergic

on membrane

Otuyo

H. B. (I 972) Acetylcholine

of guinea

choline

hrarn.

Re.\. 147, 165 169.

A. W. (1975) Loss of a-bungarotoxin

muscle

BLOCK G. A. & BILLIAR

toxin

r;ll

184, 473 475.

J. G. (1970) Dependence

ganglion

muscle

,Idenc,~mc

4409.

BLACKMAN

BUMANN

Localilatron

Bruin

cells. JI. Phys~ol.. I,clnd. 2%.

BIES~XKER G. (1973) Molecular 12. 4403

in dc\cloping

degradation

hoc.

on the synthesis

Z. W. (1974) Fate of r-hungarotoxin

.t’.y

01 Fodium-p~,tabslurn

RC.L IX. 441 4?1

Ii.

33

W. A. & RANG H. P. (1979) Studies

parasympathetic

junctions

sera and globulins.

of rat liver. J. hiol. C‘hrrn. 244. 3303

ARIMATSU Y.. St-ro

of synaptic

Brain

~:LUS S. (19771 Accelerated

gravis

ARIAS I. M.. DOLLY D. & SCHIMKE R. T. (1969) Studies reticulum

distribution

brain,

727

M. W. &

myasthenia

Subcellular

m developing

G. K. & BLOOM F. E. (1967) The formatlou

microscopic

cultured

F

and acetylcholinesterase

blocks

and fractionated

nicotinic

cobra

transmission

venom

III tile :ivIan cihar!,

on sympathellc

gdnglionrc

ganglion.

transmission.

8. 326 330

COHEN J. B. & CHAUGEI,Y J P. (19751 The chohnerglc

receptor

protein

in its membrane

environment

:t. Rec. Phurmtrc.

15, 83 103. COLQUHOIIN receptors

D. & RANG H. P. (1976) Effects in rat muscle.

CONTI-TKONCOVI

Molrc.

Phurmuc.

of the binding

8.. GOTT1 C., PAGGI P. & Rossr A. (1979) Acetylcholine

muscle of the chick:

specific

binding

srmnemi.s. Br. J. Phurmuc. 66, 33 38. C<)~MA~ C. W. & Lyucti G. S. (1976) deafferentation

of inhibitors

on new synapse

of iodinated

x-bungarol~~xm

and

effect

Reactive

formation.

York. COTMA& C. W. & NAI)L~K J. V. (197X) Reactive

In

on the

synaptic

synaptogenesis Neuronal

receptors transmission

in the

Recognitim

synaptogenesis

W.). pp. 277 271. Raven Press, New York. Ca()ss~~~i) W. J.. COWAN W. M. & ROGCRS L. A. (lY75)

adult

of the nervous

ganghon :md m the lrls

neurotoxin system.

Ihe

from

.~CJ/U :vulu

effects

of Partial

(ed BARONDES S. H.). pp. 69 108. Plenum.

in the hippocampus. Studies

in the atlary

In .%‘eitrontrl Pl~\tfc~t~

on the development

of chick

optic

autoradiographic study on the development of retina-tectal connections. Bruin Res. 91. I 23 DALE.H. H. (1914) The action of certain esters and ethers of choline. and their relation IO muwx~rInc 7‘hcv. 6. 147 1%)

to acet?)cholmr:

12, 519 535.

New

(cd. C‘c)I%fAs (‘ teCtum .I

/%w)u

IV

An (‘\I’.

Nicotinic DAME

V. N. &

KARLIN

A.

(1978) Affinity

acetylcholine

labeling

receptors

of one of two alpha-neurotoxin

from Torpedo californica. Biochemistry 17, 2039-2045. DE BELLEROCHEJ. & BRADFORD H. F. (1978) Biochemical

evidence

II

in the CNS

for the presence

binding

sites in acetylcholine

of presynaptic

receptors

receptor

on dopamin-

ergic nerve terminals. Brain Rex 142, 53-68. DE BELLEROCHE J., LUGMANI Y. & BRADFORD H. F. (1979) Evidence for presynaptic cholinergic receptors on dopaminergic terminals: Degeneration studies with 6-hydroxydopamine. Neuroscience Letters 11. 209-213. DE BLAS A. & MAHLER H. R. (1978) Studies on nicotinic acetylcholine receptors in mammalian brain. Characterization of a microsomal subfraction enriched in receptor function for different neurotransmitters. J. Neurochem. 30, 563-577. DEVEREOTESP. N. & FAMBROUGH D. M. (1975) Acetylcholine receptor turnover in membranes of developing muscle fibers.

d. Cc/l Biol. 65, 335- 358. DEVREOTES P. N. & FAMBROUGH D. M. (1976~) Synthesis of acetylcholine receptors by cultured chick myotubes denervated mouse extensor digitorum longus muscles. Proc. nutn. Acad. Sci. U.S.A. 73. 161- 164. DEVREOTESP. N. & FAMBROUGH D. M. (19766) Turnover Symp. quant.

Biol. 40,

of acetylcholine

receptors

in skeletal

muscle.

Cold Spriyy

and Hark

237-25 I.

DEVREOTESP. N., GARDNER J. M. & FAMBROIJGH D. subsequent incorporation into plasma membrane of DIAMOND J. & MILEDI R. (1962) A study of foetal and D~JI,AI Y. & SEGAL M. (1978) z-Bungarotoxin-binding

M. (1977) Kinetics of biosynthesis of acetylcholine receptor and cultured chick skeletal muscle. Cell 10, 365-373. new-born rat muscle fibers. J. Physiol.. Lond. 162. 393-408. sites in the rat hippocampus: Localization in post-synaptic cells.

Bruirl Rex 154. 167-171. DU(;GAN A. W.. HALL J. G. & LEE C. Y. (1976a) Alpha-bungarotoxin,

cobra

neurotoxin

and excitation

of Renshaw

cell:;

by acetylcholine. Brain Rex 107, 166 170. DUGGAN A. W., HALL J. C.. HEADLEY P. M., HENIIRY 1. A. & MINCHIN M. C. W. (1976h) Absence of bmding of z-bungarotoxin and cobra neurotoxin to central acetylcholine receptors-an autoradiographic study. .Yrurosc~ienw Letters 3, I ?!-- 127. EDWARDS C. (1979) The effects of innervation

on the properties

of acetylcholine

receptors

in muscle.

.~‘euro.scuwr

4,

565-584. ETEROVIC V. A. & BENUETT E. L. (1974) Nicotinic cholinergic receptor in brain detected by binding of z-[‘Hlbungarotoxin. Btochim. hiophys. Acta 362, 346-355. FAMRROL’
and axo-dendritic

synapses

GRAY E. G. & WHITTAKER V. P. (1962) The isolation

fragments

derived

by homogenization

of the cerebral

cortex:

of nerve endings from brain: and centrifugation. J. Allut. 96, 79-86.

an electron

microscope

An electron-microscoptc

study. J. ,d,,ut. studj

of cc]]

GREENI: L. A. (1976) Binding of x-bungarotoxin to chick sympathetic ganglia: properties of the receptor and its rate of appearance during development. Brain Rrs. 111, 135- 145. GREENI: L. A.. SYTKOWSKI A. J., V~CEL Z. & NIRENBERG M. W. (1973) a-bungarotoxin use as a probe for acety]choline receptors of cultured neurones. Nature, Lond. 243, 163-166. H.wLLY M. R.. BENNETT E. L. & LUKASIEWICZ R. J. (1978) Possible explanation for r-toxin failure in blocking CNS acetylcholine receptors inferred from a-toxin binding studies. Neuroscience Ahsrt. 4, 1644. HAssLER R.. CHLJNG J. W., RINNE U. & WAGNER A. (1978) Selective degeneration of two out of nine types of synapses ,,I Cat caudate nucleus after cortical lesions. E.rp/ Brain. Res. 31, 67-80.

I!

R.

E.

OSWALD

and J. A. FREEMAN

HEII)MANNT. & CHANGEUXJ. P. (1978) Structural and functional properties of the acetylcholine rrceptor m 11s purllied and membrane-bound states. A. Rev. Biochcm. 47, 317 357. HE~~MANNT. & CHANGFUXJ. P. (1979~) Fast kinetic studies on the interaction of a fluorescent ;qonist with the membrane bound acetylcholine receptor from ‘Tory& ItIcirMorutu.Eu,. J. Biochem. 94, 255 279, HEIDMANN T. & CHA~~;EUXJ. P. (1979b) Fast kinetic studies on the allosteric interactions between .tcetylchoirne receptor and local anesthetic binding sites. Eur. f. Xiockem. 94, 281 296. HEINEMANN S., BEVA?:S.. KULI.BERGR., ~1~1~s~~~~. J & R~ct J. (1977) Modulation of acctyicho~Ine receptor h) antibod) against the reteptor. Proc. rtanz. Acad. Set. c’X.1 74. X)90--3094. H~CAN P. G., MARS~~ALL J. M. & HALL. 2. W. (1976) Muscle activity decreases rate of degradation rtt ~-bun~~r~)to~ln bound to extrajunctional acetylcholine receptors. Xurur~. Lund MI. 318 330. HUNT S. & SCHMIDTJ. (1978) Some observations on the binding patterns of z-hungarotoxin in the central nervous qctcm of the rat. Brair~ Rcs. 157. 213 232. Hv?i,r S. Br SCHMIDTJ. (1979) The relationshlp of x-bungarotoxin binding activity and choEnergtc tcrmmation ulthln Ihe rat hippocampus. Nwuscienc~e 4, 585-59’ HUNT S. P. & WEBSTER K. E. (1975) The proJection of the retina upon the optic tectum of the pqeon J. c’orny. ~CWU/ 162. 433 446. KAO I. B DRACIIMAIU D. B. (1977) Myasthcnlc immunoglobulin accelerates acetylcholine receptor degradation. SCIZ,IC(‘. N.Y: I%, 527 -529. Ko C. P., BLJRTCW H. & BUNCE R. P. (1976) Synaptic transmission between rat spinal cord explants and dlssocrated superior cervical ganglion neurons in tissue culture. Brain Res. 117. 437-460. KOUVELASE. D,, DICHTERM. A. & GREENE!L. A. (1978) Sympathetic neurons develop receptors tar ~-bun~dr(~toxIil tn citro. but the toxin does not block nicotinic receptors. Brain Rex 154, 83-93. KOLIVF.LAS E. D. &. GREENEL. A. (1976) The binding properties and regional ontogeny of receptors itar ./-bungaroto~in m chick brain. Brclin Res. 113. i 11-126. LEE C. Y. (1970) Elapid neurotoxins and their mode of action. C/m. I&icoi. 3. 457-472. LEF C. Y.. CHANC S. I.... KAU S. T. & SHIX-H~X L. (1972) Chromatographic separation of the venom of Htrttr/uru\ mulricinctus and characterization of its components. J. Chromar. 72. 71 -82 Le~rz T. L. & CHESTERJ. (1977) Localization of acetylcholine receptors in central synapses. 1. C’cj/lBiol 75. 258 267. LINI)END. C. & FAMBROUGH D. M. (1979) Blosynthesls and degradation of acetylcholine receptors In rat skeletal muscles. Effects of electrical stimulation. Neuroscience 4. 527. 538. LOWEJ., MCGREGORJ., ROSENSIDNE J. & SCHMIDTJ. 11976)Solubilization of an z-bungarotoxin-bmdmg component from rat brain. Bio~~f~rn~sfr~ 15, 1522-l 527. L~JKAS R. f.. Mo~~I~~~o H. & BENNETT E. L. (1979) Effects of Theo-group modifi~tion and Ca’! on ~~8onist-s~ific ~tzte transitions of a central nicotinic acetyjcholi~e receptor. Bioc~ern~sr~~18, 2384-239s. LLKASEWICZ R. J. & BEKNETTE. L. (1978) ~-bungarotoxin binding properties of a central nurtous system nicottmc acetyicholine receptor. ~~o~~jrn. bio~~ys~ Acta 544,294-308. LUTIN W. A.. BRADY R. N., JENSEN C. F., SKENE P. & FREEMANJ. A. (1975) Preparation UIJ chara~ert7~ll~~n of horseradish peroxidase conjugated snake neurotoxin. Neuroscience Absrt. f, 630. MCGEERP. L., MCGEERE. G. & INNANEN V. T. (1979) Dendroaxonic transmission-.-l. Evidence from receptor binding of dopaminergic and cholinergic agents. Brain Res. 169, 433-441. MCQUARRIEC.. SALVATERRA P. M.. DE BLATA., ROUTESJ. & MAHLERH. R. (1976) Studies on nrcotimc acct~lcholrne receptors in mammalian brain. J. hiol. C&m. 251, 6335-6339. MAELICKEA.. FULPU B. W., KLETTR. P. & REICH E. (1977) Acetylcholine receptor. Responses to drug hmchng. .1. &of Cltam. 257, 4811 -4830. MARSHALLL. M. (1979) Subsynaptic localization of alpha-bungaratox~n binding which blocks mcotinic transmission at frog sympathetic neurons. ~~u~o~eie~~e Abs. 5.2505. MEHLIE J. P.. CHANCEUX J. P. & GROS F. (1976) Acetylcholine receptor de~adation measured hv pulse chase l~h~l~i~l~. Nururr.

Lotrd.

264,

74.- 76.

MERLE

J. P., CHANGE~.X J. P. & GROSF. (1978) Skeletal muscle acetyIchoii~e receptor. Purl~~tit)n. charactcrl~arion. and turnover in muscle cell cultures. J. hiol. Client, 253, 2882-2891. MERLIEJ. P.. SOBELA.. CHANGEUXJ. P. & GROS F. (1975) Synthesis of acetyichoiine receptor durmg dt~er~ntiat~oi~ of cultured embryonic muscle ceb. Proc. nafn. Acud.

Sci. U.S.A.

72, 4028-4032.

MII.EDI

R. & PO’ITFRL. T. (1971) Acetylcholine receptors in muscle fibers. Nature, Land 233. 594, MI. MII.EI)I R. & SZCZEPANIAK A. C. (1975) Effect of Dendroaspis neurotoxins on synaptic transmission 11)the spInal cord (11 the frog. Pror. R. SW. B 190, 267-274. MII.LCRJ. V.. LLI~CAS R. J. & BENNETT E. L. (1979) Effects of thiol modification and Ca’- on agonist-specific sliitc transitions of nicotinic acetylcholine receptor from Torpedo californica electroplax. Lge Sci. 24. 189%I!KKl ~~~~~ M. p. H. & R,@TBRY &i. A. (1979) Studies of reversible and irreversible interactions of an alkylating agonist with ~o,lt& c~f~or~~ic~acetylcholine receptor in membrane-bound and purified states. Biochemisrry 18. 1862--t867. ,MSR. N. ($976) Studies of nicotinic a~tyl~ol~n~ receptor protein from fat brain. Bi~J~~~~m. b~(~P~~~s. Actu 44d. 25: 260. MOORE: W. M. & &.AW

R. N. (1977) Studies

pu~fication. ~~~)c~~i?~l. b~op~~s. M(X)KE

Acta

498,

of nicotinic acetylcholine

receptor protein from IXt bram

11. Partial

331-340.

IV. .I. & f_,(n I\i. J. (1972) Irreversible binding of a krait neurotoxin to-membrane proteins from ccl ctectrttPl‘&s .in(t hag brain. Bi0~h~~t~.hi&p.~. RCS.Commun. 46. 20~2099.

Nicotinic acetylcholine receptors in the CNS MORLEY

B. J. &

KEMP

G. (1978) Developmental

studies of I-bungarotoxin

binding sites in mammalian hi-am.

13 .Seuro-

science Abs 4. 1656. KEMP G. E. (1980) Characterization of the nicotinic acetylcholine receptor in mammalian brain. J. in press. MORLEY B. J., LORDEN J. F., BROWNG. B., KEMP G. E. & BRADLEYR. J. (1977) Regional distribution of mcotinic acetylcholine receptor in rat brain. Brain Res. 134, 161-166. MURRAY M. (1976) Regeneration of retinal axons into the goldfish optic tectum. J. camp. Neuro/. 168, 175 1%. NORDEN J. J. & FREEMAN J. A. (1980) Quantitative ultrastructural analysis of the goldfish optic tectum following enucleation. Neuroscience Abs. 6. NORDEN J. J., &BERG A. J. C. & FREEMAN J. A. (1978) Descriptive and quantitative EM studies of the optic tectum of Xenopus following enucleation. Neuroscience Abs 4, 2048. NOZAK~ y., SCHECH~ER N. M., REYNOLDS J. A. & TANFORDC. (1976) Use of gel chromatographq for the dctcrmmation of the Stokes radii of proteins in the presence and absence of detergents. A reexamination. Biochemi.strl~ 15. 3x84 MORLEY B. Receptor

J. &

Rex,

3890.

NURSEC. A. & O’LAGUEP. H. (1975) Formation of cholinergic synapses between dissociated sympathetic neurons and skeletal myotubes of the rat in cell culture. Proc. natn. Acad. Sci. U.S.A. 72, 1955~1959. OBA~AK. (1974) Transmitter sensitivities of some nerve and muscle cells in culture. Brain Rex 73. 71-88. O’LAGUEP. H.. OBATAK., CLAUDEP., FURSHPANE. J. & POTTERD. D. (1974) Evidence for cholinergic sqnapses between dissociated rat sympathetic neurons in cell culture. Proc. natn. Acad. Sci. U.S.A. 71, 3602-3606. &BERG A. J. C. & NORDENJ. J. (1979) Ultrastructural study of degeneration and regeneration in the amphibian tectum. Brairl Rex 168. 441-455. OSWALDR. E. (1979) The nicotinic acetylcholine receptor from toad and goldfish brain. Ph.D. Thesis. Vanderbilt University. OSWALDR. E. & FREEMANJ. A. (1977) Amphibian optic nerve transmitter: ACh, yes; GABA and glutamate. no. 1Veuroscience Abs 3. 1308. OSWALDR. E. & FREEMANJ. A. (1979) Characterization of the nicotinic acetylcholine receptor isolated from goldfish brain. J. hiol. Ckem. 254. 3419-3426. OSWALDR. E. & FREEMANJ. A. (19800) The goldfish brain nicotinic acetylcholine receptor: Evidence for tMo Interconvertible y-bungarotoxin binding sites. Biochim. biophys. Acta., in press. OSWALDR. E. & FREEMANJ. A. (1980b) Degradation rate of goldfish brain nicotinic acetylcholine receptor. eBru111 Res. 187, 499-503. OSWALDR. E.. SCHMIDT D. E. & FREEMAN J. A. (1979) Assessment of acetylcholine as an optic nerve neurotransmitter In Bufo murinus. Neuroscience 4, 1129-l 136. OSWALD R. E.. SCHMIDTJ. T.. NORDENJ. J. & FREEMAN J. A. (1980~) Localization of r-bungarotoxin binding to the rctino-tectal projection of the goldfish. Bruin Res. 187, I 13-l 27. OSWALDR. E.. SOBELA.. WAKSMANG., RCIQUES B. & CHANGEUX J. P. (1980b) Selective labelling by [3H]-trimethisoquin azide of polypeptide chains present in acetylcholine receptor-rich membranes from Torpedo murmorutu. FEBS ktler.\. Ill.

29-34.

PATKI~ICJ., M~MILLANJ., WOLFSONH. & O’BRIENJ. C. (1977) Acetylcholine receptor metabolism in a nonfusmg muscle cell line. J. hiol. C’hem.252. 2143-2153. PATRICKJ. & STALLCUPW. B. (19770) Immunological distinction between acetylcholine receptor and the alpha-hungarotoxin-binding component on sympathetic neurons. Proc. nafn. Acad. SC;. U.S.A. 74, 4689-4692. PATRICKJ. & STALLCXJP W. B. (19776) a-Bungarotoxin binding and cholinergic receptor function on a rat sympathetic nerve line. J. biol. Chem. 252, 8629-8633. PFENNINGER K. H. & REESR. P. (1976) From the growth cone to the synapse: properties of membrane involved in synapse formation. In IYeuronal Recognition (ed. BARONDES S. H.), pp. 69-108. Plenum Press. New York, POLZ-TEJERA G.. SCHMIDTJ. & KAR~ENH. J. (1975) Autoradiographic localization of r-bungarotoxin binding sites in the central nervous system. Nature. Lond. 258. 349-351. RAFTERYM. A.. BLANCHARD S., ELLIOTJ., HAUTIGP.. M~QREH. P.. QUASI U., SCHIMERLIE M.. WITZEMANK V. & W[ W. (1979) Properties of Torpedo californica acetylcholine receptor. Ado. Cytopharmacol. 3, 159-182. RAFTFRYM. A.. SCHMIDTJ., CLARKD. G. & WOLCOTTR. G. (1971) Demonstration of a specific r-bungarotoxln hlnding component in E/ectrophorus electricus electroplax membranes. Biochem. biophys. Res. Commun 45, 1627 -1629. RAFTERYM. A.. SCHMIDTJ., MARTINEZ-CARRION M., MOODYT., VANDLENR. & DuGuIr~ J. (I 973) Biochemical studIcs on Torpedo calijornica acetylcholine receptors. J. supramolec. Struct. 1, 360-367. RAFTt:Rv M. A., VANDLENR. L., REED K. L. & LEE J. (1976) Characterization of Torpedo californica acetylctloline receptor: Its subunit composition and ligand-binding properties. Cold Spring Hurh. Symp. quant. Bio/. 40, 193 202. RAN’C; H. P. (1975) Acetylcholine receptors. Q. Rev. Biophys. 7. 283-399. RAWIN P. M. & BERG D. (1979) Inhibition of neuronal acetylcholine sensitivity by r-toxins from Bu,tqcrrut nltr/ticrnctus venom. Proc,. nutn. Acad. Ser. U.S.A. 76, 2072-2076. SALVATERRA P. M. & FODERS R. M. (1979) [“‘I]-a-bungarotoxin and [3H]quinuclidinylbenzilate binding In central nervous systems of different species. J. Neurochem. 32, 1509-1517. %%I-VATEKRA I’. M.. CURD J. M. & MAHLERH. R. (1977) Interactions of the nicotinic acetylcholine receptor from rat brain with lectins. J. Nrurochem. 29, 345-348. SAL.VATtRRA P. M. & MAHLLR H. R. (1976) Nicotinic acetylcholine receptor from rat brain: Soluhilization. partial purification and characterization. J. biol. Chem. 251, 6327-6334. SALvATf’RRA P. M., MAHLERH. R. & MIXIREW. J. (1975) Subcellular and regional distribution of lZSI-labeled x-hunparo-

1-l

R. E.

(%W~i.l)

and J. A. FKEI~MA’~

toxin binding in rat hrain and its relationship to acetylchoiinesterase

and choline acetyit~~nsfe~~s~

J. hoi.

C’hm, ZSU.

6469 6475. SALVAT~RRA

P. M. &r MoI)R~.W. J. (1973) Binding of i~“‘11-r-bungarotoxit~ to particulate fracttoni of rat and guinea pig brain. Biockcrn. hiophgs.Rex C’omnzut~ 55, 13I I i 31X. SCH~CTER N.. FRANUS 4.. DPUIXH P. G. & C~AZLANGA M. S. (1979) Recovery of tectal nii-c,tlnlc-choii;~crRlc receptor sites during optic nerve regeneration in goldfish. Bruin Res. 116, 57-64. !%HI:~TI:RN.. HANI)Y1. C.. PEZ~EMANTI L. & S~.HMII)~J. (1978) Distribution of r-bungarotoxin bmdmg sues in the ccnlr:rl nervous system and peripheral organs of the rat. Toxicon 16, 245-251. Sctn_ntrr:n L. S. & ELDI:FKAWIM. E. (1974) Identification of the nicotmic and muscarinic acc~\Ichuhne receptors III subcellular fractions of mouse brain, Nruropharfnacoloyp 13, 57 63. %HMII)? J. (1977) Drug binding properties of an r-hungarotoxin binding component from rat bram H&c Plrurrnrc< 13. 283-290. SCHMIDTJ. T. (5979) Movement of optic terminaIs in goldfish tectum after local pre- or post\!n‘rptic blockade of transmission. Nruroscrefrce .4hs. 5, 2 I64 Sfrr+ttr)r J. T. 6s FREEMANJ. A. (JY79) Comparative effects of ~-bungar(~toxiil on retina-tec~dl t~~lislni~s~orl in different vertebrate phyla. frrresr. Op~?~~~. Sup@. 280. SCHMIIX J. T. & FKEEMAX J. A. (1980) Eiectrophysjologjc evidence that retinotectaf synaptic tranonusston in the goldfish 15 nicotinic cholinergic. Ercrin Res. 187, 129 142. STHMII>T-NIELS~N B. K., GEPNERJ. I., TENC;N. N. H. 8r HALLL. M. (1977) Characterization of an a-bungarotoxin binding component from Drosophila n~elanogaster. J. Neurochem. 29, 1013-1029. Scltt~t:~zr- S. M., F’RANK E. F. & ~ISCHBACH G. D. (1978) Channel open time and metabohc stabtIlt! of synapttc and extrasynaptic acetylcholine receptors on cultured chick myotubes. Pror. mtn. Acad. Sci. C’.S..t. 75. 5N 52.1 SI:GEI. M., DUoAt Y. & AMSTI;RI>AM A. (1978) Distribution of an r-bungarotoxin-binding cholincrgrc nrcotinic receptor m rat brain. Brrrin Rc,.s.148, 105. 119, &TO A., ARIMATS~I Y. & AMANI)T. (1977) A glycoprotein resemblrng a peripheral nicotintc dcetylcholme reccptnr th;tt binds [‘*5]I-r-bungarotoxin in mouse brain. Neuroscience Letters 4, 115 119. SILVER J. & BILLIARR. 8. (1976) An autoradiographic analysis of [~H]~-bungarotoxin distribution rn the rat hr;nn ;tftcr intraventricular injection. J. c’eli Biol. 71.956963. Skn~rt:~M. & FUISCHtK S. (1977) Characterization of Triton X-I(~-solubilized prostag~andin f- bmdmg protem of rat liver plasma membranes. J, hi& Cheer. 252, 3689~3696. Sonrrr A.. WCRERM. 6: CHAN~;UJ~J. P. (1977) Large scale purification of the acetllchulmc receptor protein in 11s membrane bound and detergent-extracted forms from Toy& marmorata electric organ. Eur. J. Hn~chcm.80,21’; 224, Sr>I:rttR. C.. CHEN F. M.. LINIISTROM J. M., KOBAYASHI R. M. & YAMA~UKAH. J. (1977) Nicotinic cholincrgic receptors in rat brain identified by [‘z5I] Nujrr Naju siamensis x-toxin binding. Brain Res 131, 35@355 T~I(‘HRERG V. 1. & CHAIL’~EUX J. P. (1976) Presence of two forms of acetylcholine receptor wtth different tsoelectrrc points in the electric organ of Electrophoru.s elrc,rricu.\ and their catalytic interconversion in vitro. FEES Ixrfrrs 67, 264 268 THOMASW. E.. BRAI)~ R. N. & TOW~SIL J. Ci. f 1978) A characterization of x-bungarotoxin bindmg in the brain of the horseshoe crab, Limuhts polyphemus. .Art It, IIiochcrn. Biophys. 187, 53 hO. TINDALLR. S. A., KENT M., BASKIN F. & RO~~NHI:RG R. N. (t978) Nicotinic acetylcholine receptor 01 mammalian brain: .vrrju toxin binding to subcettUh.+rfractions of rat brain. .I. Srurocltrnt. 3% 859-863. Tr: A. T. (1973) Neurotoxins of animal venoms. ‘4. Rec. Broclu,nr. 42. I??.( 2% VOGEI.Z. & NIREMERG M. (1976) Localt,atron of acet~lcholine receptors durmg syI)apt~~g~nesi~m rettna. f’toc. n&b? Aead. Sci. C.S.A. 73, 1806 -1810. WADE P. D. & TIM~RAS P. S. (19X0)Whole brain and regional [ i 2~I]-alp~a-bungarotoxin binding tn developing rat. Rnritt Rrs. 181, 381 389 WANG G. K. & SCHMIDTJ. (1976) Receptors for alpha-bungarotoxin in the developing visual syslem of the chick. Elrurn RYS. 114. 524. 529. YAXLLA S. & SC.HMIU~’ J. (1976) Radioautographrc localization of [ ‘2s1)-alpha-bungarotoxin binding sttes m the retmas of goldfish and turtle. Vision Rrs. 16, 8788880. YAZCLLAS. & SCHMIDTJ. (1977) Two types of receptors for alpha-bungarotoxin in the synaptic IdFrs Of pigcOn retina Bruin Rex 138, 45S?. YOSHI~)A K. & IM\IRA H.

(1979) Nicotinic chotinergic receptors in brain synaptosomes. Rrcrtn Rrs 172. 453 439 thcrpted

2 Jrriy

I9801