Effect of α-bungarotoxin on retinotectal synaptic transmission in the goldfish and the toad

0306-4522/SO/OSOl-0929102.00/O

Neuroscience Vol. 5, pp. 929 to 942 Pergamon Press Ltd1980. Printed inGreat Britain 6 IBRO

EFFECT OF wBUNGAROTOXIN ON RETINOTECTAL SYNAPTIC TRANSMISSION IN THE GOLDFISH AND THE TOAD J. A. FREEMAN,J. T. SCHMIDTand R. E. OSWALD Departments of Anatomy, Ophthalmology and Biochemistry, Vanderbilt University Medical School, Nashville, TN 37232, U.S.A. Abstract-Alpha-bungarotoxin has been used extensively to characterize the acetylcholine receptor at the myoneural junction and in the electric organ, where it irreversibly blocks the response to acetylcholine. a-Bungarotoxin has also been employed in numerous biochemical studies of presumed acetylcholine receptors in the brain, although physiological studies demonstrating its effectiveness in blocking the response to acetylcholine are lacking, and a-bungarotoxin is ineffective in blocking the response to acetylcholine in sympathetic ganglia neurons. In-this study a-bungarotoxin was applied to the optic tectum of the goldfish and of the marine toad in dilute concentration (lo-’ M) either directly to the pial surface, or to the tectal neuropil by a micropipette. The latter method of application resulted in the rapid irreversible abolition of excitatory postsynaptic potentials generated in dendrites of tectal neurons by optic nerve activation, as shown by analysis of the laminar current source density. Responses to nonretinal (thalamic) inputs were unaffected, indicating specificity of action. Topical application of a-bungarotoxin was ineffective in the goldfish, and of considerably reduced effectiveness in the toad, due to a substantial diffusion barrier. posed by the pia-arachnoid. A mathematical method based on the solution of the differential equation describing simultaneous diffusion and chemical reaction was developed to determine both the diffusion coefficient and the association rate constant for cc-bungarotoxin from physiological measurements. We estimate the diffusion coefficient and the Stokes radius of z-bungarotoxin to be approx 1.7 x 1O-6 cm*/s and 12 A respectively, and the association rate constant to be 5.1 x IO4 Me1 s-l, in reasonable agreement with values obtained by biochemical methods. Possible reasons for the differences in effectiveness of r-bungarotoxin in blocking the response to acetylcholine at different synapses are discussed in terms of possible differences in subunit composition of the oligomer complex of the acetylcholine receptor and in its dissociation kinetics.

THE POLYPEPTIDEsnake venom neurotoxin cc-bungarotoxin (c(-BTX) has provided an extremely useful tool for the localization and biochemical characterization of nicotinic acetylcholine receptors (AChR) at the myoneural junction and in the electric organ. In these tissues a-BTX not only binds selectively, with saturation, and with high affinity and pharmacological specificity, but also abolishes agonist-induced ion translocation as demonstrated physiologically (LEE & CHANG, 1966; CHANGEUX, KASAI & LEE, 1970). This has led to the extensive use of a-BTX as a probe for the nicotinic AChR in the nervous system, where similar specific binding properties have been demonstrated in sympathetic ganglia, retina and other structures in a number of different species. Thus far, however, there have been few physiological demonstrations of the effectiveness of a-BTX in blocking cholinergic transmission at interneuronal synapses. Recent work from our laboratory showed a-BTX to be effective in blocking retinotectal synaptic transmission in the toad (FREEMAN& LUTIN, 1975; FREEMAN, LUTIN & BRADY, 1975; FREEMAN,1977). Further, CHIAPPINELLI & ZIGMOND (1978) have found that it

blocks nicotinic transmission in the avian ciliary ganglion, and MARSHALL (1979) has found that it blocks nicotinic transmission at frog sympathetic neurons. In other studies on sympathetic ganglion neurons, however, a-BTX has been shown to be ineffective in blocking cholinergic agonist-induced ion translocation (CHOU & LEE, 1969; NURSE & OILAGUE, 1975; PATRICK & STALLCUP, 1977a,b; CARBONETTO,FAMBROUGH&

MULLER, 1978; KOUVELAS, DICHTER &

GREENE,1978). The native

venom from which a-BTX is obtained consists of a number of separate components (LEE, CHANG, KAU & SHING-HUI, 1972). Recently it has been shown that several of the cc-toxin components which may occur as trace contaminants in cr-BTX, have markedly different potency in blocking synaptic transmission in chick ciliary ganglion (RAVDIN, NITKIN & BERG, 1978, and persona1 communication). In the present study, we have compared the physiological effects of highly purified a-BTX on retinotectal transmission in the goldfish and the toad, which are both believed to possess a significant cholinergic retinotectal projection (GRUBERG& FREEMAN,19756; FREEMAN,1977; OSWALD & FREEMAN,1977; OSWALD,

Abbreviations: AChR, acetylcholine receptor; garotoxin; CSD, current source density.

SCHMIDT& FREEMAN,1979; OSWALD, SCHMIDT,NOR-

BTX, bun-

DEN & FREEMAN,1980; SCHMIDT& FREEMAN,1980). 929

930

J. A. FREEMAN. J. T. SCHMIDT and R. E. OSWALI)

EXPERIMENTAL Electrophysioloyicul

PROCEDURES

methods

Goldfish (Curassius uurutus, common) were obtained from Ozark Fisheries (Richland, MO) and toads (Bufo marinus) were obtained from Mogul-Ed (Oshkosh, WI). Electrophysiological recording procedures for either the goldfish or the toad are described in detail elsewhere (SCHMIDT, 1979; FRIZMAN. 1977). Briefly, fish were anesthetized by immersion in a 0.1% solution of tricaine methanesulphonate (MS222) prior to and during surgery (exposure of the tectum dorsally and of the optic nerve in the orbit behind the eye). A less concentrated solution maintained anesthesia during recording. Toads were anesthetized by injection of MS222 (0.13 mg/g) into the dorsal lymph sac. The optic nerve was stimulated with rectangular pulses (90 V x 0.2 ms) delivered through a bipolar electrode. Stimulus artifacts were electronically suppressed (FREEMAN. 1972). Field potentials were recorded from the tectal layers via d.c.-coupled pipettes (1-2pm tip) filled with saline. Potentials were averaged and stored by a PDP-12 computer which then computed the sources and sinks of current via a one-dimensional analysis (radial to the tectal surface) of current source density using a five-point smoothing/differentiating formula to obtain the divergence of the field potentials with a minimum of noise (FREEMAN & NITHOLSON, 1975). This procedure allows quantitative assessment of the strength of the synaptic activation at the separate layers of the optic projection and is capable of resolving sources and sinks spatially separated by 25 pm or more (FREEMAN & NICHOLSON. 1975). using a lattice spacing of 50pm. Tissue conductivity in the direction normal to the tectal surface has been shown to be substantially constant across the entire tectum of another species of fish. except at the boundaries where it cannot be measured accurately (VANEGAS. WILLIAMS & FRKMAN. 1979). Thus in the present study conductivity was treated as a constant. Purificution

and administration

of‘a-hunguroto.~in

The native venom from Bunyarus multicinctus consists of a number of separate components (LEE et ul.. 1972). Among these components are several postsynaptically acting a-toxins (Peaks II and III, Fig. 1) which might differ in their physiological efficacy (RAVDIN et (I/., 1978). In order to obtain each component separately and to assure a negligible contamination by b-BTX, considerable care was taken in purifying native toxin. Lyophilized B. multicinctus venom (Miami Serpentarium) was purified by chromatography on a 1.5 x 30cm CM-Sephadex C-50 (Pharmacia) column equilibrated in 50m~ ammonium acetate buffer, pH 5.0. The column was developed with a 5OOml linear gradient of 50 mM ammonium acetate buffer, pH 5.0 to I M ammonium acetate buffer, pH 6.8 (LEE it ~1.. 1972). Peak II (Fig. 1) was pooled, diluted and rechromatographed on a CM-Cellulose C-52 (Whatman) column (I x 2Ocm) equilibrated in 0.1 M ammonium acetate. Protein was eluted with a 160 ml linear gradient of 0.1 M to 0.3 M ammonium acetate (RAVDIN et al.. 197%) yielding two separate peaks (peaks 11, and II2 of LEE rt al.. 1972). The second peak (II,) was pooled and used in subsequent physiological studies. Purity was assessed by polyacrylamide gel electrophoresis by the procedure of REISFLLI), LEWIS & WILLIAMS (1962) and sodium dodecylsulfate-urea polyacrylamide gel electrophoresis (SWANK & M~INKRES. 1971). Only one band of protein was observed with coomassie blue G-250 staining. In addition, sedimentation

Frc;. 1. Chromatography of crude Bungurus multicinctus venom on CM-sephadex C-50. The column was cquilibrated in 50m~ ammonium acetate buffer (pH 5.0). Two hundred mg of the crude lyophilized venom was dissolved in 3 ml of the equilibration buffer. After loading the column and collecting 20 fractions (3 ml each) with the equilibration buffer. a linear gradient (50m~) ammonium acetate, pH 5.0, to I M ammonium acetate. pH 7.0) was used to elute the remainder of the proteins. The arrow indicates the point at which the gradient was begun.

equilibrium analysis in a Beckman model E analytical ultracentrifuge revealed that the preparation was monodisperse (R. E. OSWALD, R. G. HAMMONDS & J. A. FREEMAN, unpublished observations). Similarly, peak III (Fig. I) was also separated into two components (III, and III,) using the above procedure. Solutions for application to the brain were prepared in varying concentrations ranging from IO-’ to IOmh M in fish or toad Ringer solutions, pH 7-7.4. z-Bungarotoxin and other nicotinic agents were administered either by topical application (in Ringer’s solution above the tectum) or by low-pressure. volumetric injection through a micropipette (8- 1Opm diameter tip) inserted into the tectal layers (SCHMIDT & FREEMAN. 1979). In.jections of the Ringer solutions through this apparatus generally had no effect on the potentials or the sources and sinks when the pressure did not exceed 150 mm Hg.

RESULTS Justijcution source

for

den.sit~~

the

use

of

one-dirnensionui

current

unalysis

In a previous systematic study of the current source density (CSD) analysis technique (NICHOLSON & FREEMAN, 1975; FREEMAN & NICHOLSON,1975) it was shown that in general, components of the CSD must be measured in all three spatial variables. However. if the neuronal elements being analyzed consist of a large population undergoing synchronous activation, the CSD may in some circumstances be accurately determined by measurement of the component in a single dimension only (NICHOLSON& FREEMAN, 1975, Appendix II). In the tectum of both goldfish and toad, optic nerve fibers are distributed in fairly homogeneous sheets located in sharply defined layers. Such planer fiber arrays can be expected to possess symmetry in two dimensions. The optic fibers, distributed parallel to the tectal surface, make synapses predominantly upon tectal neuronal dendrites that

a-Bungarotoxin and retinotectal transmission

;;;;

*

..

931

._

,.. q---j

FIG. 2. Three-electrode array recording for determination of the circumferential (parallel to the optic fiber beam) sources and sinks of current generated by stimulation of optic nerve (average of 10 stimuli).

On the left are the waveforms (negative upwards) recorded from the central electrode. Calibration pulse: 1mV, 2 ms. The arrow marks the position of the stimulus. (In this and subsequent records the stimulus artifact was suppressed electronically [FREEMAN,19753.) On the right are the sources (upward deflections) and sinks calculated for both the circumferential (heavy traces) and vertical directions (lighter traces). Depths are given beside each trace.

are oriented normal to the tectal surface. Thus the majority of postsynaptic currents of interest will be expected to flow in current loops oriented normal to the tectal surface and to be detectable by onedimensional current source-density analysis in the radial direction. Postsynaptic currents in the horizontal direction would be expected to cancel, due to the synchronous activation of optic fibers and the large population of postsynaptic elements thus activated. The majority of optic fibers travel in the horizontal plane so that their currents will be confined to that plane, The slowest fibers conduct at 0.5 m/s (as can be seen in Figs 4-6, described below) so that, over the interelectrode spacing used here (50,~m), the difference in latency of arrival is at most 0.1 ms. This is not resolvable at the sampling rates used by the analog to digital converter (e.g. 256 sample points for a sweep of IOOms gives a time resolution of approx 0.4ms for the ‘worst case’, that of the slowest fibers). Thus, horizontal presynaptic CSD components should be negligible. In order to determine experimentally whether a one-dimensional analysis was justified on the basis of the above considerations, we measured the individual

components of the CSD in the goldfish tectum, using two different methods. In two fish, a three-microelectrode array was used to sample field potentials generated by optic nerve stimulation: potentials were recorded at successive points of a regular threedimensional lattice extending across the entire depth of the tectum. In a third fish, a single microelectrode was advanced in 5Osm increments through the tecturn parallel to the tectal surface, at a fixed depth in each penetration (measurements were made over the flattest part of the tectum). Recordings taken with a three-electrode array showed virtually no sources and sinks of circumferential (i.e. parallel to the tectal surface) synaptic current at each depth. Figure 2 shows the case for the direction parallel to the beam of the optic fibers. For that case the ratio of vertical to circumferential sources and sinks was 3OO:l (computed by numerical integration and averaging over all depths). Other experiments showed that the circumferential components perpendicular to the fiber beam were 500-fold smaller than the vertical ones. Thus circumferential currents appear to cancel during homogeneous activation, allowing one-dimensional analysis to be used as expected.

.I.

932

A. FKEI:M~N.J. T.

SCHMID?

FIG. 3. Laminar analysis of responses m the goldfish optic tectum to stimulation of the contralateral optic nerve. Control records. (A) Computer averaged extracellular field potentials (rt = 20 stimuli/ record): (B) corresponding one-dimensional current source-.density records; (C and D) effect of microinjection of 2 ~riof 1 x to-’ M r-BTX. Records were obtained 1h after injection (which occurred over a 30 min period). Note almost complete abolition of postsynaptic potentials (C) and sources and sinks (D). with relatively little decrement of presynaptic response. compared to A and B. Calibration pulses: 2 ms, 2 mV. Negativity and current sinks plotted upwards.

Eflect of r-BTX itz the goldfish

the same (1.5-2 m/s; SCHMIDT, 1979). on r~~ino~~~~u~ s~~lupti~ t~u~f.~~?~jssjoi~ are nearly Figure 3 shows waveforms and source-sink profiles

Cobalt filling of the optic nerve fibers shows projections to the tectum in three prominent layers (SCHMIDT, 1979): a thick upper band (with a thin superficial satellite band) that fills the superficial grey and white layers (7%180pm deep), and two sparse deeper bands in the central grey (about 250 pm deep) and deep white (about 330 hrn deep) layers. The upper band contains two populations of optic fibers having conduction velocities of S and 3 m/s, whereas the conduction velocities of the fibers in the lower two bands

from a control penetration. Two prominent synaptic components, which fuse to form a large superficial negative wave (Fig. 3A), produce sinks at approx 100 and 150pm (Fig. 3B. ‘1’ and ‘2’) within the broad superficial grey and white zone. These current sinks are associated with prominent superficial and deep current sources. indicated by the vertical dotted lines in Fig. 3B. A delayed negative synaptic wave develops deeper in the tectum. This negative component is associated with current sinks located at a depth of

ff-Bungarotoxin and retinotectal transmission 250pm to 350 pm (Fig. 3B, ‘3’) corresponding to postsynaptic depolarization elicited by the two deeper bands of optic innervation, whose terminals appear to overlap at this depth (SCHMIDT, 1979; OSWALD, SCHMIDT,NORDEN & FREEMAN,1980). When r-BTX was placed in Ringer’s solution above the tectum there was no substantial change in the waveforms or sources and sinks, even after several hours at concentrations up to lo’-3 M. However, when the a-BTX solution (IO-’ M or 10.. * M) was injected under the pia, there was a rapid and irreversible diminution of the synaptically activated waveforms. Substantially similar results were obtained from all ten fish used in the study. Figures 3C and D show the field potentials and sources and sinks obtained 1 h after 2 ~1 of a 10.. ’ M solution were infused over a half-hour period in the same fish. Note that all three components were virtually abolished, but that the early deflections due to the compound potentials of (presynaptic) optic fibers were only minimally affected. The largest presynaptic component, which was the easiest to measure, maintained 90% of its previous amplitude. The other presynaptic components appeared to be unchanged. On the other hand, the postsynaptic waves were abolished; only the earliest maintained about 10% of its previous amplitude. This synaptic blockage by c&TX was not reversible for periods in excess of 4 h. By comparison, a control injection of physiological Ringer’s solution through the micropipette at the same location, prior to the injection of a-BTX produced no significant reduction of any of the pre- or postsynaptic waveforms. We also tested several other nicotinic agents for their effect on retinotectal synaptic transmission. These are the subject of a separate study (SCHMIDT & FREEMAN,1980) and will only be mentioned here. Acetylcholine, injected at 10m4M, greatly diminished the amplitude of all three responses, presumably by cellular depolarization and receptor desensitization. BW 284C51, an anticholinesterase, prolonged and diminished all three waves. Curare (10m4M) blocked all three waves but at millimolar con~ntrations enhanced and prolonged the slower waves. By contrast, atropine, a muscarinic agent, has no significant effect on retinotectal synaptic transmission, even at 1O- 3 M. The LY-BTXused in the results presented in this study corresponds to fraction II, of LEE et al. (1972). In a preliminary set of experiments, we compared the effects of other toxin components obtained from further fractionation of the components shown in Fig. 1. Fractions III, and III,, which are also cc-toxins, were both found to be of nearly equal potency to type II, in abolishing postsynaptic responses to optic nerve stimulation. Fraction II,, containing several enzymes (LEE et at., 1972) also produced a pronounced response decrement at a concentration of 1O-7 M, but this effect was reversible over a period of 1 h, unlike the essentially irreversible effect of type II2 a-BTX (SCHMIDT& FREEMAN,1980).

Effect of a-BTX in the toad

on retinotectal

933 synaptic transmission

In the toad, retinotectal synapses from several different functional classes of ganglion cells distribute in three major compact layers (GRLJBERG& FREEMAN, 1975a; and FREEMAN,1977). Stimulation of the optic nerve results in the sequential activation of five prominent sets of negative potentials. Representative field potentials from one of the four animals used in this study are shown in the three-dimensional perspective display of Fig. 4A. The corresponding sets of sources and sinks revealed by current source-density analysis are shown in Fig. 4B, which provides far greater resolution of the electrical activity elicited by optic nerve stimulation. Figure 5 (heavy traces) shows the control field potentials and CSDs, before the test injection of Ringer’s solution, elicited by stimulation of the contralateral optic nerve (1 stim./2 s, 2 x threshold). Figure 5 (light traces) shows the corresponding set of control field potentials and CSDs obtained I5 min after injecting toad Ringer’s solution beneath the pial surface through a micropipette (2pi injected over a 10 min period at a depth of IO0 pm). The microinjection procedure itself did not interfere with synaptic transmission, as shown by the close similarity between the two sets of records. Both sets of field potentials contain five prominent negative deflections also seen in the three-dimensional display obtained from another animal in Fig. 4. The CSD records reveal five corresponding prominent sets of current sinks. The earliest sink (‘I’, Fig. 5 CSD) is maximal at 250-300 pm and consists of an initial presynaptic component, followed by a larger postsynaptic component. These sinks are produced by the presynaptic volley and subsequent postsynaptic dendritic depolarization from a class of rapidly conducting myelinated (‘off’) fibers making excitatory synapses onto tectal neurons whose somas are located in the deeper tectal layers (FREEMAN,1976; 1977). A small source at a depth of 350pm accompanies the presynaptic sinks, suggesting that the ‘off’ fiber terminals are distributed predominantly perpendicular to the tectal surface. Presynaptic components of the more slowly conducting optic nerve fibers are not as readily apparent in this one-dimensional analysis, suggesting that they are oriented predominantly in a plane parallel to the tectal surface. The second set of sinks (‘2, Fig. 5, CSD) occurs at a depth of 05O~m, and is produced by depolarization of the most superficiai optic nerve fibers and of the dendrites upon which they synapse. The source for this current component comes from deeper regions of the dendritic membrane, extending down to 2OOpm. (The correlation of sources and sinks with different functional ganglion ceH types is the subject of another study, and will not be dealt with further here.) The major current sink (ra’ and ‘3b’, Fig. 5, CSD) also consists of two components, maximal at 100 and 15Opm, respectively. Corresponding sources are supplied by regions of the mem-

934

J. A.

F‘KLII.MAN.

J. T.

kHMlI>T

and

R. I.

OSWALD

FIG. 4. Three-dimensional perspective display of response of toad optic tccttim to stimulation of the showing live prominent sets of negative potentials; (B) contralateral optic nerve. (A) Field potentials, corresponding current source density waveforms, showing underlying sets of sources and sinks producand sinks plotted upw,ards. ing the potentials shown in A. Ncgativlt)

brane traversing the entire tectum and probably represent the activation of neurons whose somas are located in the periventricular cell layers and which have radially oriented dendrites. A second superficial set of sinks (‘4’. Fig. 5. CSD) develops, due to the activation of a more slowly conducting po~ul~t~~~n of unmyelinated retinal alferents and the dendrites upon which they make excitatory synapses. This is followed by a smaller sink (‘5’. Fig. 5, CSD) at a depth of iOO/~rn. This pattern of sources and sinks was very similar in all animals, although the relative amplitude of different components was found to vary slightly depending on the position of the recording track with respect to the area of tectal representation of the area centralis (FREEMAN,1977). In contrast to the goldfish, topical application of r-BTX to the pial surface of the toad tectum does result in a decrement in postsynaptic act&y following optic nerve stimulation, as previously reported (FREEMAN,1977). The time of onset of the effect is

greatly shortened. however. by microinjection of the toxin directly into the tectal neuropil. The CSD records of Fig. 6 show the time course of the effects of z-BTX on retinotectal transmission in the same animal whose control records are shown in Fig. 5. In this ~xp~ritnelit. 7 lil of x-BTX (1 x IO- ’ M) were steadily iqected over a period of I min through a micropipette tip IOOpm below the tectal surface. There is a progressive diminution of all five major postsynaptic components, leading to a complete block of synaptic transmission, which had not returned by 5 h after the z-BTX injection, when the experiment was discontinued, The rapid deep presynaptic component (‘1’. Fig. 6) remains essentially unaffected. The microinjection of a-BTX at other sites in this animal. and in three other animals. led to a similar abolition of all five major postsynaptic components. but had a negligible effect on the amplitude of the presynaptic ‘off component, suggesting ;L postsynaptic site of action tsec Discussion).

a-Bungarotoxin

FIELD

and retinotectal

transmission

935

CSDs

POTENTIALS

-

3ooAJ

35o*r

4oocl

FIG. 5. Superimposed control records of laminar responses to optic nerve stimulation recorded in the toad tectum. Heavy and light traces show, respectively, responses before and I5 min after the injection of 2 ~1 of toad Ringer’s solution at a depth of 100 pm. Five prominent sets of postsynaptic responses are seen in both the field potential records (left) and in the corresponding CSD records (right). The injection procedure caused no significant change in the responses. Average of ten stimuli at each depth. Calibration pulses on field potential records: 5 ms, I mV, negativity (and CSD sinks) plotted upwards.

It is interesting to note the time course of synaptic blockade at different depths after microinjection of wBTX. This is related both to the binding kinetics of a-BTX with receptors, and to the diffusional mobility CONTROL

CSDs

5

MIN

of the toxin as it spreads away from the locus of injection. Measurement of the time course of synaptic blockade at different depths thus provides a potentially useful means of characterizing the kinetics of 15

60

MIN

I__

MIN

---

4

FIG. 6. Etfect of a-bungarotoxin on retinotectal synaptic transmission in the toad tectum. Progressive decrement of CSD responses to optic nerve stimulation is seen at the indicated times following micropipette injection of 24 of a-BTX (I x IO-’ M) at a depth of 100pm beneath the tectal surface. The relative times at which each of the five major CSD components become decremented by the same percentage were used to calculate the diffusion coefficient and association rate constant for a-BTX, as described in the Appendix. The small presynaptic sinks and associated sources due to firing of the myelinated ‘off’ fibers remain unaffected at 60min post injection (records shown at three times the amplitude of the other records), whereas all five major postsynaptic sinks are essentially abolished. Sinks plotted upwards.

936

J. A.

FR~ENAN.J.

T.

SCHMIDT and R.

toxin receptor binding and of estimating the size of x-BTX from its diffusion coeflicient. as described below. In order to estimate these parameters, a simple model describing the rate of removal of toxin in the presence of simultaneous diffusion and immobilization by a pseudo-first order reaction with receptor molecules was developed, as described in the Appendix. WC assumed that diffusion occurred radially outward from the tip of the micropipette. In Fig. 6. where the toxin was injected at a depth of IO0 inn. the first components to be decremented arc those sinks located nearest to this depth. followed successively by a decrement of those sinks located progressively further away. By measuring the difference in times at which diRerent components had decreased by 50’!, (see Appendix) we obtained an upper limiting value of 1.72 _t (7.064 x IO “cm’& (n = IO) for the diffusion coefficient D of r-BTX through the neuropil. This value is in reasonable agreement with the value of I.56

x IO- ” cm’4

obtained

biochemically

by measur-

ing the approach to sedimentation cqttilibrium in an analytical ultracentrifuge (R. E. OSWALD, R. G. HAMMONDS 52 J. A. FREEMAN. in preparation). Using the relationship D = ~T~(~~~~~)(Cot+?i & EDSALL. 1965) were I, is Boltzman’s constant, P{ is the viscosity of water at 20°C and r is the Stokes radius of a spherical particle. ae arrive at a rough estimate of r = I 1.9 A for r-BTX. The fact that r-BTX does not penetrate the goldfish pia arachnoid suggests that this membrane presents an efl’ective diffusional barrier to even very small molecules. This might be attributed to a smaller ‘pore size’ of goldfish pia, or to differences in the chemical composition of the pial membrane. Also using the methods described in the Appendix, we calculated an apparent association rate constant for z-BTX with receptor of 5.1 + 0.26 x IO4 M.s~- ’ (II = IO). This is in reasonable agreement with the value of 1.35 x IO4 measured biochemically in synap-

E.

OSWALD

tosomal preparations of toad brain (J. A. FREEMAN& R. E. OSWALD, in preparation). In order to determine whether the abolition of retinotectal synaptic transmission in the toad might be due to some non-specj~c effect of the toxin affecting all synapses and not just cholinergic transmission, we measured the postsynaptic activity produced in response to stimulation of other tectal afferents. before and after microinjection of r-BTX. Figure 7 shows the current source-density records computed in response to sti~luiation of the thalamic neuropil, in the region of the nucleus of Bellonci, which activates a thalamotectal pathway (TRAC.HTENRNG& INGLI:, 1974). There is a prominent early sink located at a depth of 200 pm which is preceded by a clearly visible presynaptic component. A second sink extending from the surface to 100 pm develops several milliseconds later. and has a source located radially beneath it, which evidently cuts into the earlier. deeper sink. The records (obtained 30 min after injection of 3 pl of 1 x IO-'M r-BTX) are very similar to the control records, and show no evidence of either pre- or postsynaptic decrement. We conclude that the decrement of retinotectal transmission seen in Figs 3 and 6 represents a specific effect of r-BTX on cholinergic transmission. DISCUSSION

Before considering the main results of the present study. which suggest that r-BTX blocks retinotectal transmission in both the goldfish and the toad, it is worthwhile considering possible sources of artifact in the experiments reported here. Loc~lli~arior~ 01 cwwnt sowccs und sinks. The first question is whether the technique of one dimensional current source-density analysis we employed is sufficiently accurate in localizing current sources and

oooc,

25OU 3ooL(

FIG. 7. Laminar current source-density records in the toad optic tectum produced in response to stimulation of ipsilateral thaiamic input (nucleus of Bellonci). (A) Control records: (B) corresponding records obtained at same sites 30 min after microinjection of 3 ~1 of r-BTX (I x IO-’ M). The toxin produced no significant change in response, indicating a specificity of action on retinal and not thalamic inputs. Calibration: horizontal bar. 2 ms. sinks plotted upwards.

~-Bungarotoxin and retinotectal t~nsmission sinks to permit definitive statements to be made con-

cerning the underlying synaptic events. As shown in the results of Fig. 2, the major CSD components elicited in the tectum by synchronous activation of optic nerve fibers occur in the direction vertical to the tectal surface, that is, parallel to the radially oriented dendrites of tectal neurons. These results confirm the expectation derived from theoretical considerations (NICHOLSON& FREEMAN,1975) that circumferential currents generated synchronously in a large population of neuronai elements possessing symmetry in two dimensions should cancel. The presynaptic CSD components associated with the arrival of the optic nerve volley in goldfish are very small, suggesting that the terminals are distributed predominantly parallel to the tectal surface (i.e. at right angles to the recording axis). The same is true for the slower presynaptic components in the toad tectum. There is. however, an appreciable presynaptic component associated with the most rapidly conducting ‘off’ fibers, which presumably distribute in a more radial direction before making synaptic contact with tectal neurons. A very similar set of sources and sinks to that shown here (Figs 4, 5 and 6) is also obtained using a full threedimensional CSD analysis (FREEMAN, 1977; and unpublished). Thus it appears likely that our CSD records faithfully reflect underlying synaptic events. Purity of the preparations of’ z-hungarotoxin. A second question concerns the purity of the a-bungarotoxin derivatives used in this study. RAVDIN et at. (1978, and personal communication) have demonstrated that components of peak III (Fig. 1) can block cholinergic transmission in cultured neurons from the chick ciliary ganglion, but that peak II2 (c(-BTX) is not effective. The use of high concentrations of r-BTX preparations contaminated with trace amounts of peak III (also an a-type toxin) is potentially capable of blocking synaptic transmission, indicating the need for the use of concentrations of z(-BTX in the same range as the equilibrium dissociation constant (8 nM for the toad, J. A. FREEMAN& R. S. OSWALD,in preparation; and 1 nM for the goldfish, OSWALD& FREEMAN.1979) and the use of highly purified tt-BTX preparations in judging the efficacy of r-BTX in blocking synaptic transmission. (As we discuss later, the final tissue concentration of a-BTX here was actually reduced from 10 -.’ M to approx 5 x 10e9 M by several factors.) Also, the purity of the preparation was confirmed using three inde~ndent techniques (two polyacrylamide gel systems and sedimentation equilibrium analysis). Thus, the effects observed in this study can with reasonable certainty be ascribed to c(-BTX (peak II,) rather than to trace contamination either by other r-type toxins (peak III components) or by r-toxin (peaks IV-VI, Fig. 1). Site of action of a-bungur~toxit~. A third consideration concerns the site of toxin effect. It has recently been reported (SCHWARTZ,AXELROD, FELDMAN & AGRANOFF, 1979) that c(-BTX (conjugated with rhodamine) binds to optic nerve fibers from the goldfish,

937

although the binding in unfixed tissue is very low and the type and purity of the toxin employed was not specified. On the other hand LUTIN,JENSEN,SKENE& FREEMAN(1975, and unpublished) found no evidence for presynaptic o(-BTX receptors in the neuropil of the toad tectum at the electron microscopic level using horseradish peroxidase-conjugated c(-BTX. It seems unlikely that the abolition of retinotectal transmission described here is due to a presynaptic effect of a-BTX. First, there is little if any decrement of the presynaptic components visible either in the field potential records (Fig. 3) or in the CSD records (Fig. 6). Second, neither acctylcholine antagonists, such as d-tubocurarine nor agonists such as nicotine alter the amplitude of the presynaptic optic nerve field potential components in the goldfish tectum (SCHMIDT& FREEMAN,1979). Were acetylcholine agonists to activate cholinergic receptors located on presynaptic terminals, one would expect the amplitude of the extracellular field potentials and currents either to decrease or increase, depending on whether agonist binding resulted in depolarization or hyperpolarization of the terminals. Third, application of 10m8 M cr-BTX (the same fraction II, as used in the present experiments) to neurons in slices of goldfish tectum maintained in vitro results in rapid and complete abolition of intracellularly recorded depolarizing responses produced by microiontophoretic application of acetylcholine (FREEMAN,1979b). These considerations are discussed more fully elsewhere (SCHMIDT& FREEMAN,1980). We conclude that the site of action of cr-BTX is postsynaptic, presumably on nicotinic cholinergic receptors located on tectal neurons that are monosynaptically contacted by optic nerve fibers. This conclusion is supported by recent findings that show (1) a precise correlation of a-BTX binding sites and optic nerve fibers using [‘2s1]a-BTX radioautography combined with horseradish peroxidase and/or cobalt filling of optic nerve terminals (OSWALD, SCHMIDT,NORDEN& FREEMAN,1980); (2) a dramatic loss of [‘251]a-BTX binding sites following optic nerve section (OSWALD,SCHMIDT,NORDEN& FREEMAN?1980) which is accomplished by the removal by glial cells of postsynaptic membrane specializations (presumably containing postsynaptic acetylcholine receptors) seen under the electron microscope (J. J. NORDEN,unpublished observations); and (3) a selective abolition of retinotectal synaptic transmission caused by a variety of nicotinic acetylcholine antagonists (SCHMIDT8z FREEMAN,1980). Additionally, it has been found that the optic fiber layers contain high levels of acetylcholinesterase and choline acetyltransferase in the tecta of both the goldfish (OSWALD, SCHMIDT,NORDEN& FREEMAN,1980) and the toad (GRUBERG& FREEMAN,197%; OSWALD& FREEMAN, 1977; and OSWALD, SCHMIDT& FREEMAN,1979); and that the toad optic nerve contains significant levels of acetylcholine and choline acetyltransferase. as shown by pyrolysis gas chromatography/mass spectrometry (OSWALD,SCHMIDT& FREEMAN,1979).

938

J. A.

FKEMAY.J. T. SCHMIDT and R. E. OSWALI)

Specificity of the effixt of r-hunyarotaxin. A fourth consideration concerns the specificity of the toxin effect. As previously mentioned, we employed a concentration of a-BTX (peak II, of LEE ct ul., 1972) in

the same range as the equilibrium dissociation constant for a-BTX measured bi~hemically in both toad and goldfish synaptosomal fractions. The actual tissue concentration of x-BTX is likely to be substantially less than the concentration of a-BTX used to fill the injecting micropipettes, due to binding of x-BTX to the glass surface. Using scintillation counting of [‘“‘I]c(-BTX (10m7 M)ejected from mi~ropipettes in a similar fashion to that described here, we have found (SCHMIDT & FREEMAN, 1980) that the concentration of the solution ejected from the tip had dropped to 2 x 10-S M, while the actual tissue concentration was around 5 x 10e9 M, using a value of 30’?4,for the extracellular votume as calculated by the use of [“HIsucrose (FREEMAN & OSWALD,1977. and unpublished). Thus there is likely to be a 20-fold dilution of toxin concentration in the tissue. These considerations, taken together with the fairly low background binding of [‘2SI]~-BTX in synaptosomal fractions of the goldfish tectum (OSWALD& FREEMAN.1979) suggest that the predominant effects of r-BTX reported here can be attributed to specific binding to r-BTX binding proteins and that r-BTX binding proteins

and nicotinic cholinergic receptors are probably similar if not identical entities (i.e. share common binding sites} in both goldfish and toad tectum. The results shown in Fig. 7. in which r-BTX has little if any effect on tectal responses to thalamic stimulation. further suggest that a-BTX is acting at specific synaptic sites.

Given the adequacy of our recording methods, the purity and specificity of PBTX. and the selectivity of

its effects on post synaptic elements possessing nicotinit cholinergic pharmacology, we conclude that the results of the present study provide strong physiological evidence for the efficacy of rx-BTX in blocking synaptic transmission in the amphibian and teleost central nervous system in low concentrations. a-BTX was ineffective in blocking synaptic transmission in goldfish when it was applied topically to the surface of the tectum but it was dramatically effective when applied directly to the synaptic regions via microinjection from a micropipette. By contrast. r-BTX applied to the surface of the pia-arachnoid of the toad tectum was effective in blocking’ synaptic transmission, although the time required was much longer than that following localized microinjection. The pia evidently represents a substantial diffusion barrier to r-BTX (~W-78~; LEE, 1972) which ought to be taken into account in subsequent studies. We calculated a value for the Stokes radius of a-BTX of approximately l2A (assuming it to be a spherical molecule), using the methods described in the Appendix. This vaIue, which should only be taken as ap-

proximate but which is probably close to the true value, suggests that the goldfish pia represents a very effective molecular sieve. It is noteworthy that the mathematical techniques used to estimate the diffusion coefficient of PBTX can also be used to estimate the association rate constant, from physiological as opposed to biochemical measurements. Similar methods should prove useful to obtain biochemical parameters from physiological measurements in other systems. The accuracy of the values obtained (which compare closely to those obtained biochemically) depend on the correctness of the assumption that an equal percentage decrement of response at two tectal sites following z-BTX application represents an equivalent percentage of r-BTX-receptor binding at the two sites. This in turn is based on the assumption that receptor density and binding properties as welt as re~ptor-mediated ion transfocation are similar at the two sites. Currently experiments are in progress to measure these parameters at the cellular level, using intracellular recording and noise-spectral analysis of synaptic potentials recorded from neurons in tectal slices (FREEMAN.1979a.h). It should be pointed out that the correct use of the methods described in the Appendix requires accurate determination of the locations (i.e. the radial distances from the locus of injection) of synaptic or other physiological effects produced by the injected substance. To rely on field potential measurements alone to determine the locus of synaptic events can be very misleading, and one should use the technique of current source-density analysis which provides far more accurate resolution.

Several studies have raised interesting questions about the identity of a-BTX binding components in the nervous system. For example. a-BTX has been found to be ineffective in abolishing acetylcholineevoked excitation of sympathetic ganglion cells (CHOU & LE. 1969: N~~RSE& CYLAGC~.1975; CARBOVETTO et a!.. 1978; K~LWELAS c/t~1..I978). CAKl%ONi?TTO et ai. (1978) recently found that the a-BTX receptor from cultured chick sympathetic ganglia neurons had a higher sedimentation velocity in sucrose gradients than muscle AChR, and suggested that z-BTX and acetylcholine bind to different sites on sympathetic neurons. Similarly, PATRICK & STALL~LZ (1977a) reported that antisera to the eel x-BTX AChR complex do not recognize the x-BTX--receptor complex from a cultured rat sympathetic neuron tumor cell line having presumed nicotinic synapses, nor does r-BTX abolish cholinergic agonist-induced Na’ Rux. They conclude that the r-BTX-receptor and the functional AChR are different entities both in these cells and in other neurons as well. It is possible that r-BTX and acetylcholine bind to sites on different subunits of the functional AChR. Using a selective photoaffinity label, WITZEMANN&

r-Bungarotoxin

and retinotectal transmission

RAFTERY(1977) report that the AChR from Torpedo consists of four separate poly~ptide subunits, and that the structural arrangement of these subunits, which appear to be different for membranebound versus solubilized receptor, determine their binding affinities for the label. Further, RAFTERY, VANDLEN,REED& LEE(1975) have shown that c(-BTX binds to two populations of receptor sites in purified AChR from 7: californica, only half of which bind acetylcholine or other cholinergic ligands with high affinity. Thus the cl-BTX receptor might be an integral part of the AChR oligomer, but occur on a different physical (or functional) subunit than the acetylcholine binding site(s). Failure of a-BTX to block the response to a~tylchoIine might then occur if the structural arrangement of the subunits, or of the binding of r-BTX on them, were such that a-BTX did not shield a critical binding site for acetylcholine. It may be that the subunit composition of the AChR oligomer varies in different neuronal cell types such that binding sites for a-BTX (or other nicotinic ligands) overlap those for acetylcholine (BLJLGER,JUIAN-JUIAN,HINDY, SILBERSTEIN& HESS, 1977; ROBBINS,ANTOSIAK,GERDING & UCHITEL,1977) for some synapses but not others. If this idea is correct, the dissociation rate of the a-BTX-receptor complex may serve as an indicator of the eflicacy of a-BTX in blocking synaptic transmission. Both goldfish (OSWALD & FREEMAN,1979) and toad (OSWALD & FREEMAN,1977) tectal r-BTX binding proteins consist of two kinetically distinguishable components, one of which has a slow dissociation rate (t l/2 = 44 h for the goldfish and 127 h for the toad at 20°C). The slow rate of complex dissocic~~~rnic~

939

ation is similar to that observed with or-BTX in muscle (BROCKES & HALL, 1975), and with Nuja naja sianlensis a-toxin (a similar long-acting a-neurotoxin) in electroplax (MAELICKE & REICH, 1976) where a-toxins are physiologically effective, in contrast to the rapid dissociation rate in sympathetic ganglion receptors, where a-BTX is ineffective. It is important to determine the identity of cx-BTX binding proteins currently being studied biochemically at other central nervous system sites. In particular, physiological studies are needed in order to determine whether other cr-BTX binding proteins in the central nervous system can be functionally equated with AChRs, or whether the two proteins should be considered as inde~ndent macromoiecules (see also HUNT & SCHMIDT, 1979). If the latter proves to be the case, the possibility that the c(-BTX binding protein might serve as a specific marker for cholinergic synaptic membranes in the CNS independently of the AChR, or that the cr-BTX binding protein might be involved in the regulation or stabilization of synaptic connections (CHANGEUX & DANCHIN, 1976; FREEMAN,1977), makes the precise identification and characterization of this membrane component a subject of considerable interest. Ackao~~led~e~ents-This study was supported by NIH Research Career Development Award EY70240 and National Eye Institute Grant EYOl117 to J.A.F. and NIH Postdoctoral Fellowship NS05437 to J.T.S. We thank Drs J. BARACHand J. WIKSWOof the Department of Physics

for helpful comments on the manuscript, and MS MARGARETBARBand MS J. SCHMIDTfor typing same.

REFERENCES BROCKES .I.P.

& HALLZ. W. (1975) Acetyl~holine receptors in normal and denervated rat diapbra~ muscle---II. Comparison of junctional and extrajunctional receptors. ~~~c~~~~~sr~~ 14, 210&2106. BULGERJ. E., JUIAN-JUIANFu. L., HINDYE. F., SILBERSTE~NR. L. & Hess G. P. (1977) Allosteric interactions between the membrane-bound acetylchoiine receptor and chemical mediators. Biochemistry 16, 684692. CARBONETTO S. T., FAMBROUGH D. M. & MULLERK. J. (1978) Nonequivalence of ix-bungarotoxin receptors and acetylcholine receptors in chick sympathetic neurons. Prof. nafn. Acud. $5. U.S.A. 67, 1241-1247. CHANGEUX J. P. & DANCHINA. (1976) Selective stabilization of developing synapses as a mechanism for the specification of neuronal networks. Nature, Land. 264, 705-7 12. CHANGEUXJ. P., KASAI M. & LEE C. Y. (1970) Use of a snake venom toxin to characterize the cholinergic receptor protein. Proc. nafn. Acad. Sci. U.S.A. 67, 1241-1247. CHIAPPINELLI V. A. & ZIGMONDR. E. (1978) a-Bungarotoxin blocks nicotinic transmission in the avian ciliary ganglion. Proc. natn. Acad. Sci. U.S.A. 75, 2999-3003. CHOU T. C. & LEEC. Y. (1969) Effect of whole and fractionated cobra venom on sympathetic ganglionic transmission. Eur. J. Pharmacol. 8, 326-330. COHN E. J. & EDSAL J. T. (196.5)Proteins, Amino Acids, and Peptides p. 402. Hafner, New York. CRANKJ. (1957) The mathematics of Dz~us~on.Oxford University Press, London. FREEMANJ. A. (1972) An electronic stimulus artifact suppressor. E~eetroeneeph. clin. ~europhys~o~. 31, 170-172.

FREEMANJ. A. (1976) Patterns of convergence of afferents onto tectal neurons in Bufo marinus revealed by intracellular recording. Sot. Res. I/is. Ophchal. 6,91. FREEMANJ. A. (1977) Possible regulatory function of acetylcholine receptor in maintenance of retinotectal synapses. Nature, Lond. 269, 218-222. FREEMAN, J. A. (1979a)Intracellular responses and receptor localization of neurons in slices of goldfish tectum. Sot. Res. I/is. Ophthal., in press. FREEMAN J. A. (1979b) Dendritic localization and density of acetylcholine receptors in single cells in slices of goldfish tectum. Neuroscience Abs. 9, 2495.

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FREEMAN J. A. & LL!TIN W. A. (1975) Use of x-bungarotoxin in the histological and elcctrophysiological identification of cholinergic synapses in the optic tectum of the toad Btcfo ~uri~z~.~. Am. Sot. ~~~{r~~~~~~i~l. 132, 143. FRIZMAN J. A.. LUTIN W. A. & BRADY R. N. (1975) Possible molecular mechanisms associated with r&no-tectal synapse formation in &c/b marinus, revealed by the use of labelled snake neurotoxin. Nrurosciertce 4h.s 5, 1.18. FREEMAN J. A. & NICHOLSON C. (1975) Experimental optimization of current source-density technique: Application to anuran cerebellum. d. Neurophysiof. 38, 369-382. FR~LMAI’ J. A. & OSWALD R. E. (1977) Extracellular K+ kinetics and synaptic tr~~nsrn~ssi~~ll in the ~irnphibi~In optic tectum. Pro<. iift. Cong. Physiol. Sci. 13. 239. GKIIIIEHG E. R. & FREEMANJ. A. (1975~) Optic projections of the toad (Bufo mclririus) as seen wrth autoradrography. M.f.7: Quurt. bog. Rep. 116. 262- 266. GRXWRCI E. R. & FREEMANJ. A. (197%) Acetylcholine esterase activity and acetylcholinc synthesis m the optic tectum of the toad B&r marinus: Probable identification of an optic nerve neurotransmitter. M.1.7: Q~c~rf. Pray. Rep. 116, 266-273. HUNT S. & STHMIDT J. (1979) The relationship of a-bungarotoxin binding activity and cholinergic termination within the rat hippocampus. Neuroscirnce 4, 585-592. KWWLAS E. D.. Df(.fiTER M. A. & GREENE L. A. (1978) Chick sympathetic neurons develop receptors for ~-b~ln~rotoxin in ritro. but the toxin does not block nicotinic receptors. Brain Rex 154, 83--93. Lr:r: C. Y. (1972) Chemistry and pharmacology of polypeptide toxins in snake venoms. ,4. Rev. Phurmuc~. 12. 265. LFI. C. Y. & CHANG C. 0. (1966) Modes of action of purified toxins from elapid venoms on neuromuscular transmission. Mtwr. Ifzsr. Bmmtrm. Simp. intrrnnc. 33, 555. LEE C. Y.. CHAW S. L.. KAC’ S. T. & SHING-HLG L. (1972) Chromatographic separation of the venom of Rtcngarzts multicinctus and characterization of its components. J. Chromat. 72, 7 I-82. Lur~lc; W. A.. JFNSENC. F.. SKENE P. & FREEMAN J. A. (1975) Preparation and characterization of horseradish peroxidase conjugated snake neurotoxin. Nruroscirncr Ahs 5, 630. MCLENI*;AN H. (1956) The diffusion of potassium. inulin, and thiocyanate in the extraccllular spaces of mammalian muscle. Biuchim. hiophys. acts 21, 472-48 I. MAI;LICKI. A. & REICH E. (1976) On the interaction between cobra r-neurotoxin and the acetylcholinc receptor. Cold Spring Hurh. Symp. quent. Biol. 40, 23ll236. MARSHAL.L L. M. (1979) Subsynaptic localization of r-bungarotoxin binding which blocks nicotinic transmission at frog sympathetic neurons. N~l~rflsci~~tcl, Ahs. 5, 743. NITHOLSON C. & FREEMANJ. A. (1975) Theory of current source-density analysis and detcrmirxtion of conductivity tensor for anuran cerebellum. J. Neurophysiol. 38, 356368. NKHOLSW C.. PHILLIPS J. M. & GARDNER-MEDWIN A. R. (1979) Diffusion from an iotophoretic point source in the brain: role of tortuosity and volume fraction. Brtiirt Rrs. 169, 580 584. N~~KSEC. A. & O’LAGU~: P. H. (1975) Formation of cholinergic synapses between dissociated sympathetic neurons and skclctal myotubes of the rat in cell culture. Proc. nutn. Acad. Sci. U.S.A. 72, 1955 1959. OSWALD R. E. & FRI:EMAN J. A. (1977) Amphibian optic nerve transmitters: ACh. yes: GABA and glutamate, no. .‘Vr,rlrosc,ic,rl~,c~.4hs 3, 1308. OSWALD R. E. & FREEMAN J. A. (1979) Characterization of the nicotinic acetylcholine receptor isolated from goldfish brain. J. hi&. Chem. 254, 3419-3426. OSWALD R. E.. SCHMIDT D. E. & FREEMAN J. A. (1979) Assessment of acetylcholine as an optic nerve neurotransmitter in Bufo marinus by the use of pyrolysis gas chromatography-mass spectroscopy. Nrurnsciencv 4. I I29 I 136. OSWALD R. E., SCHMIDT J. T., NORDEN J. J. & FREEMAN J. A. (1980) Localization of ~-bLingarotoxin binding sites to the goldfish retmotectal projection. Brain Res. 187, 113-127. PATRICK J. & STALL.~I!P W. B. (19770) Immunological distinction between acetylcholine receptor and the I-bungarotoxinbinding component on sympathetic neurons. Proc. natn. Acad. Sci. U.S.A. 74,4689-4692. PATRH‘K J. & STALLC’IIPB. (1977h) a-Bungarotoxin binding and cholinergic receptor function rn a rat sympathetic nerve line. J. hi&. Ckw. 252, 8629 -8633. RAFTFRY M. A., VANDLEN R. L., REED K. L. & LEE T. (1976) Characterization of Torprdo ca&vnica acetylcholine receptor: Its subunit composition and ligand-binding properties. Cold Spring Hurh. Symp. quant.Biol. 40, I93- 202. RAVIXN P.. NITK~N R. & BERG D, (1978) z-Bungarotoxin

binding

sites and acetylcholinc

receptors

in chick ciliary ganglion

neurons

in culture. ~~~~~rosc~~}~c~~ Ahs 4, 1904. RI:ISF~:I.I) R. A., Ltw~s U. J. & WILLIAMS D. E. (1962) Disk electrophoresis of basic protcms and pcptides in polyacrylamide gels. Nccture, Land. 195, 281-283. Ronnlxs N.. ANTOSIAK J., GERDING R. & UCHITEL 0. (1977) Nonacceptance of innervation h) mnervnted neonatal rat muscle. f)rc. Biol. 61. 166 176. STHMII)T J. T. (1979) The laminar organization of optic nerve hbres in the toctum of the goldfish. Prrje. Roy. Sot,. B. in press. SCHMIIJT J. T. & FREEMANJ. A. (1980) Electrophysiologic evidence that retinotectal synaptic transmission in the goldfish is nicotinic cholinergic. Brain Res. 186. S~RW~KT~ M.. AX~:LRO~ D.. FI;I.DMAN E. L. & AGHANOFF B. W. (1979) Histo~tloresce711 id~l~tiiic~~ti[~t~of ~-b~in~rotoxin binding sites in the goldfish visual system. Nruroscience Ahs 9, 1020. SWANK R. T. & M~INKRES K. D. (1971) Molecular weight analysis of oligopeptides by alcctrophorcsis rn polyacrylamide gel with sodium dodecyl sulfate. Anul. Biorhem. 39, 462-477. THhcliTt NHI.R(; M. C. & IN(;LE D. (1974) Thalamo-tectal projections in the frog. Bruin Rc.s. 79. 419 4.W.

941

x-Bungarotoxin and retinotectal transmission

VANEGASH., WKLIAMSB. & FREEMAN J. A. (1979) Responses to stimulation of marginal fibers in teleostean optic tectum. Expl Brain Rrs. 34, 3355347.

WITZEMANV. & RAFTERYM. A. (1977) Selective photoaffinity analogue. Biacke~n~srr~ 16. 586225868.

labeling of acetylcholine receptors using a cholinergic

(Accepted 4 November

1979)

APPENDIX Methods to determine the diffusion coeficient ation rate constant of a substance undergoing d@tsion and chemical reaction

and associsimultaneous

In the experimental situation to be modeled, a diffusabie substance (e.g. I-BTX) is injected via a micropipette into a medium (e.g. the optic tectum) containing a fixed distribution of receptors with which the substance undergoes a chemical reaction of the form C(r, t) + R(r, t) 2

where M* (mols) is the effective amount of diffusing substance injected at r = t = 0. (We assume that the injected substance remains confined to the extracellular volume, which comprises a fraction a of the total volume. so that M* is related to the actual amount rejected, M, by M* = M/U (NICHOLSON et al., 1979). Substituting A4 into Al (with k, = 0) and integrating we obtain R(r, t) = R,(r)exp[~

CR(r, t)

erfc(-$=+=)]

(A3

A2

where R,(r) is the initial mean concentration (mols/liter) of binding sites (e.g. receptor molecules) at distance I from the point of injection. Equation A5 provides the desired approximating analytic expression for the spatial and temporal distribution of unbound receptors following an instantaneous injection. (If the injection time is prolonged with respect to the time Wr, t) = --.._ (Al) required for diffusion and binding, or if the amount -k,C(r, t)R(r, t) + k2CR(r, t) = ff$f!. I3 injected per unit time varies, then R(r, t) must be obtained from the convolution of equation A5 (impulse response) The binding of n-BTX in both the toad and the goldfish tectum is essentially irreversible (OSWALD & FREEMAN, with the function describing the injection process,) Expressions for both D and k, can be obtained from AS by 1977; 1979), so we shall set kz = 0. Equation Al gives the measuring the times t, and t2 after injection at which, retime rate of change of concentration due to chemical reacspectively, the response amplitudes at two positions rl and tion. For a medium that is isotropic and homogeneous rZ, decrement by some criterion amount (say 50%) from the with respect to diffusion, the time rate of change of concencontrol amplitudes measured at t = 0. We assume that tration due to diffusion is equal percentage decrement of response at two or more ?C(r, t) locations is produced when equal percentages of receptors --= D*V* C(r, f) iit have been bound. From AS we obtain

where C(r, r), R(r, t). and CR(r, t) represent respectively, the concentration (mols/liter) of diffusing substance, receptors, and bound complex at time r (s) and distance r (cm) from the point of application; k, (M.s)-’ and k2 (s-l) are the respective association and dissociation rate constants. The kinetic equations describing this reaction are

where V2 is the Laplacian operator, and D* is the diffusion 4nD* (In OS)r, 4nD* (In O.S)r, -k, =i coefficient (cm*/s) in the complex medium (related to the diffusion coefficient in free solution, D, by D* = D/l’, M*erfc(*) = hI*erfc(2*’ (A6) where 11’ is a tortuosity factor related to the increase in average path length which the diffusing substance encounters in the complex medium (NICHOLSON, PHILLIPS Equation (A6) can be simplified and rearranged to give: & GARDNER-MEDWIN, 1979; MCLENNAN,1956). For a system undergoing both diffusion and chemical reaction, the time rate of change of concentration is given by the sum of equations Al and AZ: qqy

= D*V* C(r, t) - kl C(r, t) R(r, t).

(A31

It is desired to find an analytic solution for R(r, I) and from this to determine both D and k,. Although equation A3 is nonlinear and thus not amenable to analytic solution, it is possible to obtain a close approximation by the method described by CRANK(1957; section 8.5). in which the solution to the simplified diffusion equation (A2) is substituted into equation Al to obtain a solution for R(r, I). For an instantaneous point source in an infinite medium, the solution to equation A2 is given by CRANK (1957; equation 3.5) C(r, f) =

M* __I_

8(rrD*ty

e - .~,4LF,Lt

644)

Equation A7 can be readily solved numerically, graphicaliy, or by successive approximations with a hand calculator, to obtain D*, since r,, rZr rr and t2 are known. Once D* is determined, k, can be determined from equation A6. It should be noted that since it excludes the effect of kr, approximating equation A4 leads to an overestimate of C(r, t), and thus the computed value of I)* will be expected to be somewhat larger than the actual value, providing an upper bound. In order to determine D from D*, we used the value 01= V,/V, = 0.3 measured in the toad tectum using [3H]sucrose to label the extracellular space (FREEMAN& OSWALD.1977 and in preparation), where Vs and VT refer to the extracellular and total tectal volume, re-

J. A. FREEMAN. J. T. SCHMIDT and R. E. OSWALD

941 spectively.

The tortuosity ;,=,

(modified

+;[“,“!j(,

factor

was estimated

_a)=

from MCLENNAN. 1956. eq. 61.

from

1.19

It is interesting to note that equation A6 can be used to determine the amount of substance M liberated from the injection source if k, and D are known. This provides a potentially useful means for determining how much substance is liberated iontophoretically from a micropipette.