Chemically Induced ION Channels in Nerve Cell Membranes

CHEMICALLY INDUCED ION CHANNELS IN NERVE CELL MEMBRANES By David A. Mothers* and Jeffery 1. Barker Laboratory of Neurophyriology National institute of Neurological and bmmunlcative Disorders and Stroke Notional Institutes of Health Betherdo, Marylond

..... I. Introductionand Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... 11. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........ A. Fluctuation Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Voltage J u m p Relaxation . . . . . . . . . . . . . . . . . . . ................... C . Extracellular P a t c h c l a m p . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . A . Invertebrate Nerve Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Vertebratecentral Neurons ........................ . . ...... ..... C . Vertebrate Autonomic Ganglion Neurons . . . . . . . . . . . . . . . . . . . . . . . . ..... IV. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

2 2

6

a

10 10 18

2a 31

32

I. Introduction and Scope

The macroscopic response of chemically excitable nerve and muscle cells to agonists usually consists of brief changes in membrane current lasting milliseconds to seconds, During the past decade, our understanding of the mechanisms by which agonists produce such effects has improved dramatically. This progress may be largely attributed to the application in this field of three biophysical techniques with increased resolving powers: fluctuation analysis, voltage j u m p relaxation, and the extracellular patch clamp. These methods, which will be described in this article, have revealed that the macroscopic effects of agonists reflect, in at least some cases, the summation of many individual microscopic membrane events of rather well-defined mean lifetime and amplitude. *Present address: Department of Physiology, Faculty of Medicine, University of British Columbia, Vancouver, British ColurnbiaVh;?’ 1W5, Canada .

I INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOI. 2’3

Copyright 0 1982 by Academic Press. Inc All riRtics 01 reproduction in any fbrm resxved. ISBN 0-12-306823-9

2

DAVID A. MATHERS AND JEFPERY L. BARKER

This advance in the resolution ofavailable experimental methods has proved important in two areas. First, the physical nature of membrane processes that underlie the flow of agonist-induced currents is now amenable to direct, quantitative investigation. Second, many classes of clinically important drugs, including local anesthetics, muscle relaxants, convulsants, and barbiturates, have been found to alter the properties of microscopic current pulses induced by particular agonists. In many cases it has proved possible to correlate such effects with the macroscopic action of the drug in question, thereby providing a mechanistic explanation for the mode of action of several pharmacologically important substances. The purpose of this article is to show how application of fluctuation analysis, voltage jump relaxation, and extracellular patch clamp methods has increased our knowledge of the action of agonists and other drugs at the membrane of nerve cells from both vertebrate and invertebrate preparations. At the time of writing, the majority of studies using the three previously mentioned techniques have been performed on skeletal muscle fibers, for reasons of historical precedent and experimental convenience. This field has been treated in several comprehensive reviews (Colquhoun, 1975,1979; Rang, 1975; Katz and Miledi, 1977; Colquhoun and Hawkes, 1977, 1981; DeFelice, 1977; Neher and Stevens, 1977). Reference will be made to data obtained from muscle fibers when comparison with results from nerve cells seems appropriate. The use of these three biophysical methods in the study ofvoltage-dependent conductances in electrically excitable nerve membranes has previously been extensively discussed (Stevens, 1972; Verveen and DeFelice, 1974; Conti and Wanke, 1975; Neher and Stevens, 1977) and also lies outside the scope of this article. Finally, analysis of membrane phenomena associated with stimulation of sensory receptors by light, movement, and other physical means will not be discussed here, as the transduction processes linking stimulation with altered membrane current flow are at present unclear.

II. Methods

A. FLUCTUATION ANALYSIS Katz and Miledi (1972) noted that the depolarization produced by acetylcholine (ACh) at the frog neuromuscular junction is associated with the appearance of minute fluctuations in membrane voltage. This “ACh noise” could not be attributed to technical sources and appeared therefore to reflect events underlying the activation of postsynaptic receptors by acetylcholine. Using statistical

3

CHEMICALLY INDUCED ION CHANNELS

techniques previously used in the study of electrical noise in communications systems, Katz and Miledi (1972, 1973, 1977) analyzed the voltage fluctuations induced by ACh and obtained the first quantitative description of the elementary processes associated with the stimulation of the postsynaptic membrane by agonists. Anderson and Stevens (1973) enhanced the power of this fluctuation analysis technique by using a voltage-clamp circuit to measure membrane “current noise” induced by ACh in place of the “voltage noise” studied in earlier work. This approach avoids loss of the higher frequency components of ACh-induced noise caused by the filteringeffect ofthe membrane time constant. Because most studies are now carried out using this modification, the theoretical discussion that follows will be given in terms of membrane current rather than membrane voltage. The analysis of agonist-induced current fluctuations is approached by making several simplifying assumptions about the membrane processes involved. It is postulated that the elementary event triggered by a successful interaction between the agonist and its receptor is the transient opening of an ionic channel, the conductance of which can assume only the values 0 (closed) and y (open). Furthermore, in the presence of agonist, transitions of this channel between the open and closed states are held to be Poisson processes (Fig. 1A). Therefore, the time intervals during which the channel is in the open state will be exponentially distributed with a time constant 7 , which is the mean channel open time. It is assumed that the macroscopic current Z induced by the agonist reflects the summation of a number N of channels of this type operating independently of one another (Fig. 1A). Then the mean value of I, will be given by

f = Npi where p is the probability of a channel being open and i is the current that flows through a single open channel. The variance of I, u*,will then be uz = Npi*(1 - p

)

Hence

u V f = Npi2(1 - p)/Npi= i(l - p ) At low agonist concentrations, it is evident from the dose-response curve that

p < < 1 . At this “low concentration limit,” the ratio a 2 / ~ w i ltherefore l directly yield an estimate of i (Lecar and Sachs, 1981). The variance of Z may be

calculated as ui - u i , where u i and represent the variance of membrane current fluctuations seen before and during agonist action, respectively (Fig. l B). The value of i will depend on the electrical driving force on the ionic species that flows through opened channels. This may be calculated as where is the clamp potential and the equilibrium potential of agonist action. Using

v,

4

DAVID A . MATHERS AND JEFFERY L . BARKER

B

C

FIG. 1, (A) The upper group of traces shows the activity of 10 computer-simulated channels. Each channel undergoes transitions between open and closed states as a result ofa Poisson process. All 10channels operate independently of one another but have the same amplitude and averagelifetime in the open state. The bottom trace shows the summed activity of these 10 channels. A fluctuating signal is produced; it reflects moment-to-moment variation in the number ofopen channels present. (B) Typical observations made during the application of fluctuation analysis to the study of synaptic channels in a biological membrane. Under voltage-clamp conditions, application of an agonist for the period indicated by the horizontal bar results in a change in DC membrane current I (double-arrowhead vertical bar). In addition, the variance of membrane current is seen to increase from u: prior to agonist to u', during agonist. This increase is readily apparent in the AC current trace, which represents acondenser-coupled,amplified version ofthe DC current record. The additional variance is assumed to reflect moment-to-moment changes in the number of synaptic.channels opened by the agonist. (C) Kinetic properties of these channels can be estimated from the power spectral density (PSD) of the agonist-induced current fluctuations. It is assumed that a simple kinetic scheme of the type used to generate the simulated channels in Aalso controls the operation of the synaptic channels. Under this assumption, the PSDofthe biologicalcurrent noise is expected to be of Lorentzian form (C, smooth curve). The mean open time of the agonist-induced channels can then be calculated from the half-power frequency f, (arrow) of that Lorentz curve that affords the best fit to the observed spectral points. For further explanation, see text.

CHEMICALLY INDUCED ION CHANNELS

5

Ohm’s law, the following expression can be obtained for the mean conductance

y of a single channel

y

=

[(o:, - o;)/I]/(Yc- V,)

Temporal properties of current fluctuations induced by agonists can be assessed using the methods of fluctuation statistics. The principal mathematical techniques employed are the autocorrelation function

A-w and its Fourier transformation, the power spectral density function

s ( f )=

~ J c ( Tc o) s ( 2 ~ d~~ )

In both cases, it is assumed that the agonist-induced noise represents a random process x ( t ) whose mean parameters are time independent. The autocorrelation function determines the product of the value of this function at time t and its value after a delay T. The average of this product reflects the waveform of the underlying elementary event generating the noise signal. In the case of rectangular current pulses with exponentially distributed lifetimes, the expected autocorrelation is a simple exponential with time constant 7corr= 7 , the mean channel open time (Neher and Stevens, 1977). The power spectral density of agonist-induced noise yields essentially the same estimate of r as the autocorrelation function. The predicted power spectrum for the model of channel noise described earlier is the Fourier transform of an exponential, the so-called Lorentzian curve of equation S(f) = S(O)/[ 1 (j/fC)>‘] where S(0) is the spectral intensity at zero frequency, f is frequency, and f, is the half-power frequency at which S(f) = S(O)/2 (Fig. 1C). Furthermore, at low agonist concentration, the mean channel open time is related to f, by r = (27rfC)-l (Neher and Stevens, 1977). Although the power spectral density is defined as an improper integral, an accurate estimate of its form can be made using finite digital processing methods if a number of conditions are met. The most important of these is the Nyquist Sampling Theorem, which states that the signal must be sampled at a rate at least twice as fast as the highest frequencies of interest (Bendat and Piersol, 1971). Prior to digitalization, it is imperative that steep cut-off analog filters be used to limit the bandwidth of the noise records at the low- and high-frequency ends. This reduces distortion of the spectrum by low-frequency artifacts and by highfrequency noise folded back into the frequency range of interest by a process known as “aliasing” (Bendat and Piersol, 1971; Dionne, 1981a).

+

6

DAVID

A.

MATHERS AND JEFFERY

L. BARKER

It should be noted that an estimate of the single-channel conductance can be made from the power spectrum using the relation

y

=

S(0)/[2 17(

c;r - y ) ]

for double-sided noise spectra (Anderson and Stevens, 1973). This estimate will exceed that obtained by the variance-to-mean ratio method described earlier if a significant fraction of the total noise variance lies beyond the low-pass filter frequency employed. If this is the case, estimates of y obtained using the variance method must be corrected by dividing the calculated value by (2/7~)tan-' (&lfr), wheref,, is the low-pass filter setting (Colquhoun et al., 1977). Fluctuation analysis has been used in the study of cholinergic synapses in frog (Katz and Miledi, 1972; Anderson and Stevens, 1973), mouse (Dreyer et al., 1976b), rat (Colquhoun et al., 1977), and human muscle (Cull-Candy el al., 1979). Cholinergic transmission has also been investigated using the technique in cultured muscle cells (Sachs andLecar, 1973,1977) and in nerve cells in invertebrates (Ascher et al., 1978a,b) and vertebrates (Ascher et al., 1979). Fluctuation analysis also has been applied to the study of membrane channels induced by putative amino acid transmitters in invertebrate muscle fibers (Crawford and McBurney, 1976; Anderson et al., 1978; Dude1 et al., 1980; Mathers, 1981) and in cultured mouse spinal neurons (McBurney and Barker, 1978; Barker and McBurney, 1979a,b; Mathers and Barker, 1980a,b; Barker et al., 1981a). JUMP RELAXATION B. VOLTAGE

The voltage jump relaxation method estimates the mean open time of agonist-induced channels from relaxation currents that flow in response to a step change in membrane voltage (Adams, 1975; Neher and Sakmann, 1975). Consider the molecular transformation

where the agonist-receptor complex exists in a nonconducting state AR, and a conducting state AR*, which interconvert with rate constants and a. The equilibrium between AR and AR* is described by where Nis the total number of available receptors. The macroscopic current due to the agonist, Z, is then given as

CHEMICALLY INDUCED ION CHANNELS

7

where y is the conductance of a single channel, V, is membrane clamp potential, and Vr the equilibrium potential for agonist action. IfB and/or a is voltage dependent, application of a potential step in the presence of an agonist should result in an instantaneous ohmic change in Z,followed by an exponential relaxation of Zwith time constant 7rel(Fig. 2) given by Trel =

(a

+ b)-'

(1)

The voltage dependency of P can be assessed from the relation

p, IP I

= (zeq~'zin~t )'(7r~l'7r~l)

(2)

where P, and b are the values of at the potential after the voltage step V2and before the voltage step V , , respectively. Zcquis the equilibrium current at potential V 2 ,and Zins, the instantaneous current on stepping from V , to V2.7:el and 7Ll are the time constants for Zobtained at V2and V , , respectively. The voltage dependency of a can be obtained by combining Eqs. (1) and (2) (Neher and Sakmann, 1975). At low agonist doses, the value of fl (a concentration-dependent term) is generally assumed to be small in comparison t o a . Under these conditions, 7relis expected to be identical with 7nnoise, the mean channel lifetime obtained from noise analysis (Adams, 1975)and 7rel = 7"&

= a! -

'

TAU

FIG.2. T h e voltage jump relaxation method for determining kinetic properties of agonist-

induced membrane channels. Under voltage-clamp conditions, the membrane potential of the cell ( V ) is briefly stepped between two values. This is performed several times in both the absence and presence of the agonist. Subtracting the former responses from the latter group yields the current I, which shows how the potential step influences the current contributed by agonist-generated membrane channels. At the beginning of the voltage step, this current instantaneously assumes a new value, Zins,, which reflects the change in electrical driving force on the ions moving through open membrane channels. Provided that the channel gating process is voltage dependent, I then relaxes T h e time constant ofthis relaxation (TAU, small arrow) exponentially to an equilibrium value Iequ. provides a measure of the mean open time ofagonist-induced channels at the new membrane potential. I , represents the zero membrane current level.

8

DAVID A . MATHERS AND JEFFERY L. BARKER

Very good agreement between 7Rland rnnoise under low agonist concentration conditions has in fact been observed at cholinergic receptors in the frog end plate (Neher and Sakmann, 1975) and in Aplysia neurons (Ascher et al., 1978a). The molecular process controlled by the rate constant a could in principle involve the slow isomerization of the agonist-receptor complex from a conducting to a nonconducting state (Model A), or slow agonist dissociation (Model B). It can be shown that these two models generate different dependencies of 7xl (and 7noise)on agonist concentration. Specifically, Model B predicts an indefinite decrease of T ~with ~ , increasing agonist concentration, whereas Model A predicts a saturating relationship between these two quantities, as was in fact found experimentally at the frog end plate (Sakmann and Adams, 1978). The cholinergic agonist trans-3-(a-bromomethyl)-3’-[ a-(trimethylammonium) methyl] azobenzene (tmns-QBr) binds covalently to a point near the ACh binding sites of electroplaque cholinergic receptors (Bartels-Bernal et al., 1976). Lester et al. (1980) have shown that the kinetics, voltage sensitivity, and temperature dependence of the response of the electroplaque to trans-QBr are very similar to results obtained with reversible agonists such as carbachol. These findings also support the notion that the rate-determining process underlying ACh relaxation currents at the vertebrate end plate probably involves a molecular isomerization of the agonist-receptor complex rather than dissociation of bound agonist from the receptor. It should be emphasized, however, that this conclusion need not be applicable to all synaptic receptors. The voltage jump relaxation method has been used to study the action of cholinergic drugs at the neuromuscular junction of frogs (Adams, 1975, 1977; Neher and Sakmann, 1975; Sakmann and Adams, 1978), on the electroplaque organ (Lester et al., 1980), on the membrane of Aplysia neurons (Ascher et al., 1978a), and in bullfrog sympathetic neurons (Brown and Adams, 1980). In experiments of this type, agonists are usually applied iontophoretically or topically to the preparation. However, the voltage jump method also has been used to study the relaxation currents occurring during the action of a neurally released transmitter substance, y-aminobutyric acid (GABA). The experimental approach involved high frequency stimulation of the inhibitory nerve to produce a steady-state concentration of GABA at synaptic regions of crayfish muscle fibers (Dudel, 1978). This procedure may also prove useful in the study of synaptic events occurring in nerve cells.

C. EXTRACELLULAR PATCH CLAMP The extracellular patch clamp allows direct measurement of membrane current pulses generated by the opening and closing of individual ion channels. The method originated as an approach to the problem of obtaining adequate voltage control over the electrically excitable membrane in large invertebrate neurons

CHEMICALLY INDUCED ION CHANNELS

9

(Neher and Lux, 1969; Fishman, 1975). The central concept involved is the electrical isolation of a small patch of cell membrane by pressing a Ringer-filled glass microelectrode (0.5-6 pm internal diameter) against the cell surface (Fig. 3). The very high impedance of the membrane patch so formed greatly reduces endogenous membrane current noise, facilitating the detection of small signals. During the flow of small membrane currents ( < 10 PA) within the patch area, voltage drops occurring across extracellular pathways can be reduced to the microvolt range. The patch is therefore voltage clamped, and membrane current flow may be measured by means of a suitable, low-noise amplifier connected to the patch electrode (Neher et al., 1978). The patch clamp method allows the detection of low probability kinetic states of ion channels that may not be resolved in power spectra or in relaxation currents (Patlak eta/., 1979; Nelson and Sachs, 1979). Furthermore, the technique is ideally suited to the detailed study of receptor topography because its spatial resolution is largely governed by the area of the patch electrode opening (typically < 5 pm'). Finally, the patch clamp offers an alternative way of recording spontaneous or evoked electrical activity in nerve cells that are too small for intracellular microelectrodes to be used (Neher, 1981). The extracellular patchclamp technique has been used to study the action of cholinergic drugs on frog and rat skeletal muscle fibers (Neher and Sakmann, 1976; Neher et a l . , 1978; Neher and Steinbach, 1978) and on cultured rat

R

VCOMMAND

FIG.3 . The extracellular patch clamp method for recording membrane currents resulting from the activation of single ionic channels by agonists. A fire-polished glass microelectrode is filled with physiological salt solution containing a low concentration of agonist and pressed against the cell membrane. The activation of a membrane receptor by the agonist results in the transient flow o f a current i through a single open channel located under the patch electrode. This current flows to ground largely across the patch electrode resistance R , and the electrode-cell seal resistance R , . T h e current I measured by a current-to-voltage converter in series with R , is approximated by the relation I = i. [ R s / ( R s R p ) ] .T h e level ofbackground noise measured by thecurrent-to-voltage converter is inversely related to R,. T h e detection of single channel currents of a few picoamperes above this background noise usually requires values o f R s > 20 MQ and R s : R , > 5. T h e potential of the membrane patch under the electrode can be altered by applying voltage commands (V command) to the noninverting input of the current-to-voltage converter.

+

10

DAVID A. MATHERS AND JEFFERY L. BARKER

(Jackson and Lecar, 1979)and avian muscle (Nelson and Sachs, 1979). In addition, ion channels activated by L-glutamate, a putative excitatory transmitter, have been observed in locust muscle fibers (Patlak et al., 1979). The patch clamp technique also has been used to study ion channels induced in the membrane of cultured mammalian spinal neurons by the inhibitory transmitter GABA (Mathers et al., 1981). 111. Results

A. INVERTEBRATE NERVE CELLS 1. Action .f Glu&ma& on Neurons in the Squid Stellate Ganglion The first application of fluctuation analysis to the study of chemically induced membrane noise in nerve cells was made by Bevan et al. (1975). The action of L-glutamate (a putative excitatory transmitter at the squid giant synapse) was investigated on neurons in the squid stellate ganglion, using a single intracellular microelectrode to record membrane voltage noise in the absence and presence of the agonist. It was found that glutamate induced a depolarization (V)at the soma1 membrane, accompanied by additional voltage variance ( E 2 ) .The amplitude of the elementary voltage event underlying this response was estimated from the ratio E2/V(Katz and Miledi, 1972). An average value of 1pV at 8OC was found. The power spectral density of the glutamate-induced voltage noise could be described by a single Lorentzian term of average half-power frequency 11 Hz at 8OC, yielding a value of 15 msec for the mean lifetime of the elementary voltage pulse at this temperature. As pointed out by the authors, there are a number of reasons to question the accuracy of these measurements. First, the degree to which the additional voltage variance induced by glutamate was filtered by the membrane time constant was unknown. The precise location of the glutamate reactive sites was unknown and may have been too distant from the recording microelectrode to permit accurate measurement of glutamate-induced noise. Second, the possibility that glutamate might induce the release of transmitter from synaptic endings on the axon under study could not be eliminated. Neither of these difficulties has been encountered to a similar degree during voltage-clamp studies on arthropod muscle fibers, where glutamate is also believed to act as an excitatory transmitter (Nistri and Constanti, 1979). Experiments using fluctuation analysis have provided estimates of the mean conductance and open times of ionic channels induced by glutamate in crayfish and locust muscle fibers (Table I). As shown in Table I, estimates of the mean con-

TABLE I ESTIMATED MEANOPEN TIMES (7)AND CONDUCTANCES (y) OF IONIC CHANNELS OPENED IN MUSCLE CELLMEMBRANES BY PUTATIVE TRANSMITTER SUBSTANCES Voltage dependence Agonist Acetylcholine

Glutamate

y-Aminobutyric acid

Membrane preparation Frog Rat Snake Chick myoball Human Rabbit' heart Crab Locust Crayfish Crayfish? Locust

Effect"

Temperature ("C)

7

Wet)

(PSI

QlO

of

of

yb

71

-fd

7-1

y

Reference

E E

8.3

E

8 20 14

1 .o

1.8

32 25 25

-

Negl. NT Negl.

2.8 Negl. 3.3 NT NT NT

Anderson and Stevens(l973) Colquhoun el a / . (1977) Dionne and Parsons (1981)

E E

27 23

3 .O 1.5

39 22

-

NT Negl.

5.0 NT

NT NT

Sachs and Lecar (1977) Cull-Candy el a/. (1979)

I E E E

36 23 25 7

3.7 NT 120 15

Negl. NT N T Negl. Negl.

2.8 NT 1.5{ NT

Negl. NT pf Negl.

Nomaetaal. (1979) Crawford and McBurney (1976) Anderson et al. (1978) Stettmeier el a1 (1978)

I I I

23 23 20

9 NT 22

Rect. NT NT NT

NT NT NT

NT NT NT

Dudel ct a/ (1980)

166 1.4

1.3 2.4

5.0 33.0 4.0

+ + + +

"E, Excitatory; I, inhibitory. 'pS, Picosiemens; NT, not tested. 'A negative voltage dependence indicates that 7 is prolonged by membrane hyperpolarization. dNegl., Negligible; NT, not tested; Rect., rectification. 'Muscarinic ACh action opens K + channels. fTransition temperature present. {Two types of synaptically activated channels are present.

Cull-Candy (1981)

12

DAVID A . MATHERS A N D JEFFERY L. BARKER

ductance y of glutamate channels range from about 15 pS for crayfish muscle fibers (Stettmeier et al., 1978) to 120 pS in locust muscle fibers (Anderson et al., 1978). In the case of squid stellate ganglion neurons, an estimate of y = 30 pS at 8OC can be made, assuming an input resistance of 600k62 for these celis (Miledi, 1967) and taking the equilibrium potential for glutamate action as near 0 mV. The uncertainty of this estimate is probably too great, however, to allow comparison of this value with the more precise measurements obtained from arthropod muscle fibers. The reinvestigation of glutamate action on stellate ganglion cells using fluctuation analysis of voltage-clamped membrane responses would seem likely to provide much detailed information on the properties of a neuronal glutamate receptor. A study of this kind has, however, yet to be reported.

2 . Molluscan Neurons a. Excitatory Response to ACh. An extensive series of investigations have been performed on neurons in the pleural ganglion of the mollusc Aplysia califarnica (Ascher et al., 1978a,b; Marty, 1978; Marchais and Marty, 1979). Fluctuation analysis and relaxation methods were applied to investigate the properties of ion channels opened by cholinergic drugs in these neurons, which are excited by cholinomimetics. A two-electrode voltage clamp was used to measure membrane current. Voltage control at the cell body was judged adequate up to 500 Hz (Ascher et al., 1978a). When low concentrations of ACh were used, the power spectral density of the drug-induced current noise was well fitted by a single Lorentzian term (Fig. 4). The mean lifetime of ACh-operated ion channels, as calculated from such spectra, was independent of ACh concentration but was reduced by increasing temperature and by membrane depolarization. Estimates of the mean channel lifetime from relaxation studies were in good agreement with data from noise measurements, the ratio 7re,/7noise being 0.94. When carbachol was employed as the agonist, both rnoise and rre,shortened to about 0.8 of the values obtained with ACh, indicating that membrane channels opened by carbachol have a shorter mean lifetime than ACh-induced channels (Ascher et al., 1978a). These results are qualitatively similar to data obtained for ACh-operated channels at the vertebrate neuromuscular junction (Katz and Miledi, 1972; Anderson and Stevens, 1973; Neher and Sakmann, 1975, 1976). However, some interesting quantitative differences were found. As shown in Tables I and 11, the mean conductance y of ACh channels in Aplysiu neurons (8 pS) is appreciably smaller than values typically obtained at the frog end plate (y = 25 pS). It should be noted, however, that some uncertainty exists in the equilibrium potential for ACh action in Aplysia cells, primarily due to the presence of a Ca2+dependent K conductance that is induced during ACh application (Ascher et a!., 1978a). In addition, the value 8 pS represents an underestimate of y in +

CHEMICALLY INDUCED ION CHANNELS

'r

I

13

8

HZ

FIG.4. Power spectral density of membrane current fluctuations induced by ACh in a pleural ganglion neuron of Aplysia califmica. The spectrum is shown on linear coordinates in (A) and on double logarithmic coordinates in (B). The arrow in (A) indicates the background spectrum prior to ACh application. In (B), the observed spectrum is fitted by a single Lorentzian curve having a halfpower frequencyfc = 27 Hz. Assumingasimple model ofchannel operation, this yields an estimate for the mean open time of ACh-induced channels, T = 1/(27rfc) = 5.9 msec at 21OC. (From Ascher e t n l . , 1978a.)

Aplysia neurons, as the Tris buffer used in these studies is known to reduce the conductance of ACh channels (Dreyer et al., 1976a; Ascher et al., 1978a). It is of interest that low conductance (y 8 pS) ACh channels have also been detected in extrajunctional areas of denervated frog muscle fibers (Dreyer el al., 1976a). As also indicated in Table I, the mean lifetime TofACh-operated channels in frog (Anderson and Stevens, 1973), toad (Gage andVan Helden, 1979), and rat end plates (Colquhoun et al., 1977) decreases on membrane depolarization, the relation between T and membrane potential Vbeing well described by the equation, 7 = BeAV,when A and B are constants. To account for this behavior, Magleby and Stevens (1972) proposed that the ACh receptor protein has a net dipole whose orientation in the membrane field alters during channel opening and closing. ASP, the rate constant for channel opening, appears tobelargely independent of voltage, it is necessary to postulate that the channel-closing step is associated with the major reorientation of the receptor dipole (Neher and Sakmann, 1975). In the case of ACh channels in Aplysia neu'rons, however, 7 is not an exponential function of Vbut becomes relatively less sensitive to voltage changes at hyperpolarized potentials. Such behavior is not well described by the simple dipole model of Magleby and Stevens (1972). The data can be accounted for by

-

TABLE I1 ESTIMATED MEANOPEN TIMES ( 7 ) AND CONDUCTANCES (y) OF IONIC CHANNELS OPERATED IN NERVE CELLMEMBRANES BY PUTATIVE TRANSMITTER SUBSTANCES Voltage dependence

+

Agonist

Membrane preparation

QlO

OP

of

Effecta

Temperature ("C)

(msec)

(PS)

E

12

27

8.0

-

Negl.

5

E

22

150

NT

+

NT

NT N T

Brown and Adams (1980)

E

20

35 7

31

-

NT NT

NT NT

NT

Rang (1 981)

26

20

18

Negl, Negl.

2.8'

NT

NT NT

NT N T

7

Y

7

7

r-'

c

ACh

GABA

Aplysia ganglion Bullfrog" sympathetic ganglion Rat parasympathetic gangliond Cultured mouse spinal cord Goldfish Mauthner cell

Not stated

7.2

31

-

y 1.8

NT

1.3e

Reference Ascher el al. (1978a)

McBurney and Barker (1978) Faber and Korn (1980)

Glycine

0-Alanine

Glutamate

6

Cultured mouse spinal cord Goldfish Mauthner cell Cultured mouse spinal cord Squid stellate ganglion

26 Not stated

I

26

E

8

5.0

30

Negl. Negl.

3.0'

7.2

NT

NT

NT

NT

Faber and Korn (1980)

5.6

21.5

Negl. Negl.

NT

NT

Barker et al. ( 1 98 1a)

NT

NT

2.2 NT

15f

NT

NT

1.1'

"E, Excitatory; I, inhibitory. bNegl.,Negligible; NT, not tested. A negative voltage dependence indicates 7 is prolonged by membrane hyperpolarization 'Muscarinic ACh action closes open K + channels. 'Two types of synaptic ACh-activated channels are present. 'Mathers and Barker, 1981b. /Estimate of mean duration of elementary voltage event.

Barker and McBurney (1979a)

Bevan ct al. (1975)

16

DAVID A. MATHERS AND JEFFERY L . BARKER

assuming that permeant cations bind in a voltage-dependent manner to channel sites, thereby impeding channel closing. This model explains the observed lengthening of T at hyperpolarized potentials, and the stronger voltage dependence of T in the presence of divalent, as opposed to monovalent, cations (Marchais and Marty, 1979). It has been shown that the nature of the permeant cation species alters the kinetics of ACh channel gating at the toad neuromuscular junction (Gage and Van Helden, 1979). These results could also be accounted for by applying the voltage-dependent ion binding model developed for Aplysiu neurons. Furthermore, choice of suitable values for the parameters in this model generated a substantially exponential dependence of T on membrane potential, in accordance with the observed behavior of toad ACh channels (Marchais and Marty, 1979). It seems possible, therefore, to account for voltage dependency in synaptic channel lifetimes on the basis of two distinct models, and further work is necessary to determine the origins of this aspect of receptor function. b. ACh Antagonists. Fluctuation and voltage jump methods have also been applied to study the mode of action of several antagonists of the excitatory ACh response in Aplysiu neurons (Ascheret ul., 1978b). None of the antagonists tested (tubocurare, decamethonium, and atropine) altered the elementary current flowing through ACh-operated channels. This observation is consistent with the view that all the agents act as competitive antagonists (Katz and Miledi, 1972) but is also predicted by models involving all-or-none blockage by noncompetitive antagonists. The blocking action of tubocurare was found to be relatively greater when large doses of ACh were applied. Furthermore, the effectiveness of tubocurare increased with time during sustained ACh application and was greater at hyperpolarized membrane potentials. Voltage jump experiments revealed that in the presence of curare an approximately normal ACh relaxation current was followed by a slow inverse relaxation. The time constant of this latter relaxation was a linear function of the concentration of tubocurare used. Qualitatively similar results were obtained in the presence of hexamethonium, decamethonium, and atropine. The preceding results suggest that the agents tested exert a noncompetitive type of antagonism at ACh receptors in Aplysiu. The data are consistent with the view that the antagonists combine with the conducting state of the ACh receptor complex, converting it to a nonconducting form. A specific interpretation of this model postulates that the antagonists block open channels by binding to a site located in the ion-permeable pore itself, as has previously been suggested to explain the effects of procaine and other local anesthetics at the vertebrate neuromuscular junction (Gage, 1976; Adams, 1977). It should be noted, however, that hexamethonium does not block all curare-sensitive responses in Aplysiu

CHEMICALLY INDUCED ION CHANNELS

17

neurons. Such specificity has yet to be accounted for in terms of a pore binding model of antagonist action. c. Action of Procaine. The local anesthetic procaine profoundly alters the power spectrum of ACh current noise recorded at the vertebrate end plate. The single time constant seen in control conditions is typically replaced with a two time constant condition in the presence ofprocaine. The ACh relaxation current undergoes a comparable change in the presence of the drug (Katz and Miledi, 1975; Gage, 1976; Adams, 1977). Noise and relaxation methods have now revealed a qualitatively similar affect of procaine on ACh channels in Aplysia neurons (Marty, 1978). At low procaine concentrations (2 X lop5to M ) and high ACh concentrations, ACh relaxations and ACh noise spectra were biphasic, the two time constants being slower and faster than those measured in control solutions. At high procaine concentrations, however, only a single time constant (faster than normal) was seen in noise and relaxation measurements. The blocking action of procaine was greater at hyperpolarized membrane potentials. Of particular interest was the observation that the pattern of relaxation currents seen in the presence of procaine was dependent on the concentration of ACh applied. These results were found to be explicable in terms of a channel blocking model of procaine action essentially similar to that proposed to account for procaine action at the frog end plate. In Aplysia no evidence was found that procaine can induce a partial, as opposed to an all-or-none, blockage of open ACh channels, This result is at variance with data obtained at the frog neuromuscularjunction (Ruff, 1977). It seemsprobable, therefore, that more detailed comparison of the actions of procaine in Aplysia and frog ACh channels will reveal subtle differences between these two preparations. d. Action ofPentobarbita1. The general anesthetic pentobarbital attenuates the excitatory response of certain Aplysiu and Otala neurons to ACh (Barker, 1975; Wachtel and Wilson, 1980). Dose-response curves of the interaction between pentobarbital and ACh responses revealed that ACh responses of larger amplitude were depressed to a greater degree by pentobarbital than were responses of smaller amplitude. Kinetic analysis suggested a noncompetitive type of interaction between pentobarbital and the ACh response (Barker, 1975). Fluctuation analysis of ACh responses depressed by pentobarbital has shown that in Aplysia neurons this effect is not due to a reduction in the elementary conductance of the ACh channels, which was found to be 8 pS at 11OC-a value in good agreement with previous results for this preparation (Wachtel and Wilson, 1980). Noise spectra and relaxation currents both displayed two time constants in the presence of pentobarbital. The slower of these time constants was approximately the same as that seen in control solutions. These results have been interpreted in terms of a channel-blocking model of pentobarbital action

18

DAVID A. MATHERS AND JEPPERY L. BARKER

qualitatively similar to that originally evolved to account for the effect of barbiturates at the vertebrate end plate (see Adams, 1976). e. Inhibitory Response to ACh in Molluscan Neurons. Stimulation of the appropriate interneurons generates inhibitory postsynaptic currents (IPSCs) at the membrane of certain identified neurons in the buccal ganglion ofAplysia. Inhibition at these synapses appears to be mediated by the release of ACh and involves, at least in part, aconductance change to C1- ions (Gardner and Stevens, 1980). Under voltage-clamp conditions, the application of ACh to these cells was found to give rise to additional variance of the membrane current. Analysis of this ACh-induced noise yielded power spectra of variable form. In most cases, the data points were best fit by a double Lorentzian curve with half-power frequencies at about 9 and 50 H z for the slow and fast components, respectively. The corresponding relaxation time for the slow noise component (20 msec) was in agreement with the decay time constant for the IPSC in these cells. It is possible, therefore, that this slow component reflects the closing rate of synaptic channels activated by stimulation of the inhibitory nerve input. Whether the fast noise component is generated by additional kinetic processes occurring at this population of synaptic receptors or relates to a second, perhaps extrasynaptic, receptor type remains unclear. Assuming that the zero frequency asymptote of the power spectrum is dominated by the amplitude of the slow noise component, an upper limit of 3-16 pS was calculated for the mean conductance of the ACh-induced C1-permeable channels (Gardner and Stevens, 1980). Similar estimates have been obtained by other workers using this preparation (Simonneau et al., 1980) and from analysis of ACh-induced fluctuations underlying C1- -dependent responses in circumesophageal ganglion cells of the snail Helixaspersa (Brown et al., 1978).

CENTRAL NEURONS B. VERTEBRATE 1. In Viuo Studies

The first, and at the time of writing, only application of fluctuation analysis to the study of chemical excitability in a vertebrate central neuron in vivo was made by Faber and Korn (1980). Using the goldfish Mauthner cell, these authors compared the time course of inhibitory postsynaptic potentials (IPSPs) with power spectra of voltage noise generated by application of GABA and glycine to the cell membrane. Both GABA and glycine increase the permeability of the Mauthner cell membrane to C1- ions, and glycine is a putative inhibitory transmitter in this preparation (Diamond et al., 1973). Averaged IPSPs were found to decay exponentially with a time constant of 6.6 msec-considerably

CHEMICALLY INDUCED ION CHANNELS

19

longer than the membrane time constant in these cells (0.2-0..4 msec). Therefore, it was assumed that the IPSP should accurately reflect the time course of the underlying inhibitory postsynaptic current. Power spectra of voltage noise induced by both GABA and glycine were well fitted by single Lorentzian curves of average half-power frequency 22 Hz. It was suggested that both amino acids open ion channels whose mean duration (7.2 msec) is very similar to the time constant ofdecay of the IPSP. This similarity has led the authors to conclude that the relatively slow time course of the latter is probably determined by the slow closing rate of the synaptic channels rather than by the persistence of transmitter in synaptic clefts. Implicit in this argument is the contention that the natural inhibitory transmitter at the Mauthner cell membrane is either GABA or glycine. The similarity of the mean lifetimes of GABA- and glycine-operated channels observed in the Mauthner cell in vivo contrasts clearly with results obtained from cultured mouse spinal neurons. Using a two-electrode voltage clamp to control membrane potential, Barker and McBurney (1979a) found that the mean duration of GABA-operated channels (20 msec) was significantly longer than that of glycine-activated channels (5 msec). It is not yet clear whether this discrepancy results from differences in the nerve cells selected for study or whether it reflects the different experimental techniques used to record agonistinduced membrane noise.

2 . In Vitro Studies a. Tissue CulturedMouse Spinal Neurons. The complexity and heterogeneity of the intact vertebrate central nervous system (CNS) has led some workers to develop alternative strategies for studying chemical excitability in central neurons. Recently techniques that allow the primary culture of CNS-derived nerve cells as dissociated monolayers have been devised. Although these cultured cells lack the normal organization of neural tissue, they possess electrical and chemical excitabilities that resembles those characteristic of neurons in the intact CNS. For example, spinal cord ( S C ) cells that are derived from 13-day-old mouse embryos and maintained in culture for 2-5 months respond to iontophoretically applied GABA and glycine with an increase in C1- conductance qualitatively similar to that observed in some CNS neurons in vivo (Barker and Ransom, 1978a). Furthermore, cultured SC cells are relatively large (20-40 pm somal diameter) and are readily visualized, making possible the application of a two-electrode voltage-clamp system to the study ofagonist-induced membrane currents in this preparation. During the past 3 years, fluctuation analysis methods have been applied to quantify the elementary electrical events underlying agonist responses in these cells. b. GABA. Iontophoretic application of GABA to the somal membrane of

20

DAVID A . MATHERS AND JEFPERY L. BARKER

cultured SC cells results in the flow of a membrane current I and in the appearance of additional membrane current variance u 2 .Both l a n d a2became insignificant at the equilibrium potential for C1-, suggesting that an increased membrane conductance to this anion dominates the response of SC cells to GABA under the conditions used, The ratio a2/Iwasfound to be constant for responses of up to 7 nA, and calculation yielded an estimate of 18 p s for the mean conductance of GABA-activated C1- channels in these neurons (Table 11). Subsequent estimates of y have averaged slightly less (Mathers and Barker, 1980a,b; Barker and Mathers, 1981), but there is a marked variation in y and 7 for channels activated by GABA and other neutral amino acids in cultured mouse spinal neurons(Barkeretal., 1981a). Thevariationmay bedue to the fact that estimates were made in a population of unidentified spinal cord cells. Estimates did not vary for channels activated repeatedly over a 4-hr recording period. Power spectral density plots of GABA-induced current fluctuations were generally best fitted by a single Lorentzian term. The mean lifetime T of GABAactivated channels, calculated from these spectra, was found to be about 20 msec at 26°C. T was markedly more temperature dependent than y , decreasing as temperature increased with a Q,, of about 3 (D. A. Mathers and J. L. Barker, unpublished observations). No significant dependence of either y or 7 on membrane voltage could be detected over the -30 to -80 m V potential range (McBurney and Barker, 1978; Barker et al., 1981a). The preceding data were obtained when GABA was applied at the level of the cell body. These results may be compared with data obtained using similar methods at the membrane of the crayfish muscle fiber, where GABA is thought to mediate an inhibitory form of neuromuscular transmission (Table I). In this preparation, GABA-induced current fluctuations usually gave rise to power spectra best fitted by the sum of two Lorentzian curves, indicating the presence of two kinetic processes in most fibers. In the case of the faster noise component, q = 5 msec and y = 9 pS were calculated, whereas for the slower component, 7, = 33 msec was found (Dudel et al., 1980). Both these components probably reflect activation of channels in postsynaptic membranes, as membrane currents evoked by tetanic stimulation of the inhibitory nerve also displayed two relaxation time constants in response to a voltage step (Dudel, 1978). Futhermore, the magnitude and voltage dependence of these time constants were in agreement with predictions made by spectral analysis of current fluctuations induced by GABA. Both the fast and slow kinetic processes activated by GABA on crayfish muscle fibers are associated with an increase in membrane conductance to C1-. However, the slow component probably also involves flow ofNa and/or C a 2 + ,as its inversion potential is more positive than either Vc,- or VK+(Dudel, 1978). Interestingly, evidence has also been reported indicating cationic involvement in the response to GABA of +

CHEMICALLY INDUCED ION CHANNELS

21

cultured mouse spinal neurons (Barker and Ransom, 1978a) and in the guinea pig hippocampus (Alger and Nicoll, 1979). The ionic dependency and the kinetics of membrane events underlying these responses are not yet known. c. Glycine. The neutral amino acid glycine (like GABA, aputative inhibitory transmitter in the mammalian spinal cord) was found to open C1- permeable ion channels whose mean duration was about 5 msec and whose mean conductance was about 30 pS in cultured SC cells studied at 26OC (Barker and McBurney, 1979a; Barker et al., 1981a). The membrane channels activated by glycine are thus briefer and more highly conducting than those opened by GABA in this preparation. P-Alanine, another neutral amino acid endogenous to the CNS, activated C1- channels whose electrical properties were significantly different from either GABA or glycine (Barker et al., 1981a). Thus, each amino acid activates a C1- channel with different electrical properties, and unique transfers of charge per channel event are induced by each agonist structure. If the properties of these elementary current events determine the amplitude and time course of synaptic signals mediated by the amino acids in the CNS, as occurs at some neuromuscular preparations (Dude1 et al., 1980), then inhibitory synaptic signals with different properties should be generated by each amino acid. Whether the amino acids interact with the same receptor sites or with different populations of sites cannot be inferred from these results, as agonists of different structure are known to activate ion channels of different mean lifetimes and possibly also of dissimilar conductances, even when interacting with a presumably homogeneous receptor population, such as that found at the frog end plate (Katz and Miledi, 1973; Colquhoun et al., 1975; Dreyer et al., 1976a). However, the notion of receptors specificfor each amino acid is supported by the selective antagonism of amino acid responses in spinal neurons and primary afferent terminals by convulsant drugs (Barker et al., 1975; Nicoll et al., 1976) and by the relative lack of interaction of the amino acids in binding assays on CNS tissues (Olsen eta!., 1978). d. G A B A Analogs. A number of naturally occurring and synthetic substances structurally similar to GABA can mimic the inhibitory action of GABA when applied to vertebrate central neurons in vivo (Nistri and Constanti, 1979). These compounds, which are structural analogs ofGABA, have been shown to displace bound GABA from frozen rat brain synaptic membranes in a competitive manner. It has been inferred from these results that the analogs are capable of interacting with membrane receptors for GABA in CNS tissues (Enna and Snyder, 1977; Karobath et al., 1979; Greenlee et al., 1978). The properties of chloride-permeable ion channels opened by a variety of GABA analogs on cultured spinal neurons have been estimated using fluctuation analysis (Barker and Mathers, 1981). Some ofthese results are summarized in Table 111. The data suggest that the analogs tested activate membrane chan-

22

DAVID A. MATHERS AND JEFFERY L. BARKER

TABLE I11 ESTIMATED ELECTRICAL PROPERTIES OF C1- CHANNELS ACTIVATED BY GABA A N D ITSSTRUCTURAL ANALOGS IN CULTURED MOUSE SPINAL NEURONS" Agonist

r (msec)

~ ( P S ) ~ n (cellsy

y-Aminobutyric acid truns-Cyclopropaney-aminobutyric acid trans-4-Aminocrotonic acid y-Amino-8-hydroxybutyric acid 3-Aminopropane sulfonic acid 6-Aminovaleric acid Isoguvacine 4,5,6,7-Tetrahydroisoxazolo(5,4c]-pyridin-3-01 Muscimol Dihydromuscimol 5-Methylmuscimol

29 -16' 11 f 2 25f6 15 f 3 9*2 9*1 14 f 2

17 f 4' 17 f 4 17f3 16 f 4 19 f 3 18 f 5 19 f 3

88

5

28

18 f 3 17*3 16* 3 15 f 4

9 25

35 143 57

~~

*

11 3 65*14 57 f 16 14 f 4

7

4 6 11 4

6 6

n (0bs.y

541

40 34

51 62 21

27

~~~~~

"J.L. Barker and D. A. Mathers, unpublished observations, and modified from Barker and Mathers, 1981. Temperature, 23OC. pS, Single channel conductance in picosiemens. 'n (cells), Number ofneurons tested; n (obs.), number of spectra obtained on cells tested. dSignificantlydifferent from 7 values for all GABA analogs tested when compared on the same membrane (p < 0.001, student's t test). 'Not significantly different from y values for all GABA analogs tested when compared on the same membrane.

*

nels whose conductance is similar to that of GABA-operated channels. However, the mean lifetimes of analog-induced channels differ significantly from those of channels opened by GABA. It is not certain whether all of the structural analogs tested activate C1- channels through engagement of GABA receptors. The mean duration of membrane channels opened by an agonist is highly and significantly correlated with the concentration of that agonist required to displace 50% of bound GABA from membranes derived from the vertebrate central nervous system (Barker et al., 1981b; Barker and Mathers 1981). The correlation suggests that the biochemical and biophysical assays are measuring a common parameter that reflects the interaction of the agonists with GABA receptors. Ifwe assume that all of the agonists are acting via GABA receptors, then the results demonstrate that the kinetics of GABA receptor-coupled anionic channels depend on the structure of the agonist. Similar conclusions have been reached at cholinergic (Colquhoun et d., 1975; Dreyer et al., 1976a) and glutamate-sensitive synapses (Crawford and McBurney, 1976; Anderson eta!., 1978). Further study of C1- channel activation by different structures should provide some insight into the structural requirements for activation of C1- channels in central neuronal membranes.

CHEMICALLY INDUCED ION CHANNELS

23

e. Barbiturate-Induced Membrane Current Noise. Racemic mixtures of the barbiturate pentobarbital are used clinically as hypnotics and general anesthetics. Pentobarbital has been shown to exert multiple effects on vertebrate central neurons when studied in preparations of hemisected frog spinal cord (Nicoll and Wojtowicz, 1980) and in monolayer cultures of mouse spinal neurons (Barker and Ransom, 1978b; Macdonald and Barker, 1979). One of these actions involves a direct increase in the C1- conductance of the neuronal membrane. This effect of pentobarbital has been observed in vertebrate spinal cord cells (Barker and Ransom, 1978b; Macdonald and Barker, 1979; Nicoll and Wojtowicz, 1980) and dorsal root ganglion neurons (Nicoll, 1975). Interestingly, the barbiturate-evoked C1- conductance is blocked by the GABA antagonists picrotoxin and bicuculline, and it has been initially proposed that the “GABA-mimetic” action of pentobarbital may be mediated via GABA receptors (Barker and Ransom, 1978b; Nicoll and Wojtowicz, 1980). The effect of pentobarbital on the C1- conductance ofcultured mouse spinal neurons has been investigated using fluctuation analysis (Mathers and Barker, 1980a). The purified isomer (- )pentobarbital was used in these studies, as the (-) isomer is consistently more potent than either the racemic mixture or the (+) isomer in activating C1- conductance (Huang and Barker, 1980). It was found that, like GABA, (- )pentobarbital induces additional fluctuations in membrane current when applied to voltage-clamped cultured mouse spinal cord cells. Analysis of these fluctuations showed their power spectral density to be of Lorentzian form (Fig. 5). The results suggets that, like GABA, (- )pentobarbital generates inhibitory membrane responses by activating a population of two-state C1- channels. The half-power frequency characteristic of spectra derived from ( - )pentobarbital-induced fluctuations was 1 Hz at 25OC, some 5-fold lower than that found for GABA spectraobtained at the same temperature from the same cells. Estimates of the conductance of single channels activated by the drug did not differ significantly from estimates made for GABA-activated channels in the same membrane. Preliminary results with the patch clamp technique have also shown that (- )pentobarbital activates ion channels whose estimated conductance is similar to that of GABA, but whose mean duration is considerably longer (Mathers et a/., 1981). Benzodiazepines also activate a C1- conductance in cultured mouse spinal neurons. Fluctuation analysis of the response of the neuronal membrane to one of these drugs (diazepam) suggests that this agent activates ion channels whose conductance is similar to that of GABA channels but that remain open somewhat longer (Barker and Study, 1981). f. Potentiation of GABA Responses by Barbiturates and Benzodiazepines. Both anesthetic and anticonvulsant barbiturates are known to enhance the action of GABA in a variety of in vivo and in vitro preparations of the central nervous

-

24

DAVID A . MATHERS AND JEFFERY L. BARKER

FIG. 5. Power spectral density of membrane current fluctuations induced by CABA (upper spectrum) and (-)pentobarbital (lower spectrum) at the membrane of a single mouse spinal neuron in tissue culture. Both spectra have been normalized by dividing each spectral density point, S(0, by the zero-frequency asymptote of the spectrum, S(0). Least squares analysis shows that the two spectra are well approximated by single Lorentzian curves (smooth lines) drawn acIn the case of the GABA spectrum, the halfcording to the equation S(f)/S(O) = I/[ 1 (f4)']. power frequency f, = 4.8 Hz (arrow), whereas for (- )pentobarbital f, = 1.2 Hz.Assuming that the mean lifetime Tof the agonist-induced channels is given by 7 = 1/(2mfc), T~~~~ = 34 msec and T for (- )pentobarbital = 130 msec. Clamp potential, - 70mV. Temperature, 25OC.(Modified from Mathers and Barker, 1980a.)

+

system (Nicoll, 1975; Barker and Ransom, 1978b). Barker and McBurney (1979b) used fluctuation analysis to study the potentiation by phenobarbital of GABA responses recorded in cultured mouse spinal neurons. The barbiturate was found to shift the half-power frequency of GABA spectra to the left on the frequency axis. It was suggested that phenobarbital acts by prolonging the mean open time of GABA-induced ion channels. Further work has shown that clinically rekvant concentrations of phenobarbital are not effective in potentiating GABA responses. However, the (- ) isomer of the anesthetic barbiturate pentobarbital does enhance GABA responses in a dose-dependent manner, using concentrations effective clinically (Study and Barker, 1981). Fluctuation analysis of ( - )pentobarbital-potentiated GABA responses showed that the barbiturate causes a dose-dependent shift of the GABA spectrum to lower frequencies (Fig. 6). The results are consistent with the notion that the potentiating effect of (- )pentobarbital is due to an increase in the mean lifetime of GABA channels, so that more charge is transferred per ion channel event. The observed shift i n j was sometimes larger than that necessary to account for the increase in the amplitude of the membrane current response induced by GABA in the presence of the drug. This result suggests that (- )pen-

-CHEMICALLY INDUCED ION CHANNELS

CONTROL

25

CONTROL

1

1

1

01

.5

5

FREQUENCY 500 .2 (Hzl

50

20

200

FIG.6. Spectral analysis ofGABAresponses potentiated by diazepatn and(- )pentobarbital in the same cultured mouse spinal neuron. The cell was voltage clamped to - 60 mV and GABA applied to the cell body by pressure from a pipet containing 25 pi4 GABA. GABA was applied alone (control conditions) or in the presence of either 10 FMdiazepam or 50 p M ( - )pentobarbital. The spectra have been normalized as in Fig. 1C.The spectra of GABA-evoked fluctuations in the presence of the drugs were calculated from membrane current responses approximately twice the amplitude of the control membrane current response to GABA. Each of the control spectra are averages derived from 30,720 points; the spectrum obtained during diazepam potentiation utilized 56,796 points, and that taken during barbiturate potentiation 49,152 points. Each ofthe spectra (uneven lines) closely approximate a Lorentzian equation (smooth lines). The mean open time of GABA, is the corner freactivated channels, 7,can be calculated from the relation 7 = ( 2 ~ / ~ ) - 'wherefr quency (v)of the spectra. Theft of the spectrum derived from the diazepam-potentiated GABA response (8.3 Hz) is not significantly different from that obtained in control (7.7 Hz). The f, of the spectrum calculated from (-)pentobarbital-potentiated GABA responses (2.9 Hz) is significantly different from that characteristic of its control (7.1 Hz). Thus, r ranges from 20.7 to 22.4 msec under control conditions, does not change during diazepam potentiation ( 7 = 19.2 msec), and increases markedly during ( - )pentobarbital potentiation ( 7 = 54.9 msec). Temperature, 2 3 T . (R. Study and J.L. Barker, unpublished observations.)

tobarbital, in addition to increasing 7 , may also decrease the frequency of channel openings v as calculated from the data using the relation

v

=

as/i'T

where i is the amplitude of the elementary ion current. It is now known that pentobarbital increases the apparent affinity of GABA for its receptor and decreases the dissociation rate of GABA from its receptor (Willow and Johnston, 1980,1981). It is possible that these changes in the binding of GABA to its receptor are related to the altered kinetic behavior of GABA channels seen in the presence of pentobarbital.

26

DAVID A . MATHERS AND JEFFERY L . BARKER

Iontophoretically applied phenobarbital has been shown to increase the time constant of decay of C1--dependent synaptic currents in cultured mouse spinal neurons (Barker and McBurney, 1979b). Prolongation of these synaptic currents may involve changes in channel kinetics similar to those described earlier for barbiturate-induced potentiation of GABA responses. Fluctuation analysis has also been carried out on GABA responses potentiated by benzodiazepines (Study and Barker, I98 1). GABA-induced responses potentiated by the benzodiazepine diazepam are associated with elementary ion channel events that have the same conductance as those activated by GABA alone. In some spectra derived from potentiated responses, there is also no detectable change in f, (Fig. 6), whereas in others there is a modest shift off, to lower frequency values. The latter effect is never as marked as occurs during potentiation with pentobarbital and, when present, is insufficient to account for the increased amplitude of the membrane current response induced by GABA in the presence of diazepam. Calculations showed that v invariably increases during potentiation by diazepam. Thus, diazepam appears to potentiate macroscopic membrane current responses evoked by GABA primarily by increasing the frequency of channel events and secondarily by increasing, in some cases, the duration of a channel event. These results may account for the benzodiazepine-induced increase in the amplitude of inhibitory synaptic potentials recorded in cultured avian spinal cord cells, in the absence of any change in the time course of these events (Choi el al., 1981). Patch clamp analysis should reveal further details of the interaction between benzodiazepines and GABA receptors. g. Antagonism of GABA and Glycine Responses by Conuulsants. Several plant alkaloids and synthetic substances that cause convulsive activity in vivo antagonize the inhibitory effects of the neutral amino acids GABA and glycine in a variety of preparations (for a review, see Nistri and Constanti, 1979). The same convulsants readily antagonize C1--dependent responses evoked by GABA and glycine in cultured mouse and avian spinal neurons (Macdonald and Barker, 1978; Choi et al., 1981), and these effects have been examined using fluctuation analysis (Barker el al., 1980). The results show that picrotoxin and bicuculline depress GABA responses without changing the electrical properties of the GABA-induced channel event. Strychnine depresses glycine responses also without changing the properties of channels opened by this amino acid. Thus, these convulsants appear to be able to eliminate a population of channels in an all-or-none manner. Presumably they depress the amino acid responses by reducing the frequency of ion channel events and leave the remaining receptor-channel complexes to function in a manner indistinguishable from that occurring normally. The sites of interaction between the convulsants and the receptor-channel complexes have not yet been determined, Two other convulsants, pentylenetetrazole and penicillin, appear to change

CHEMICALLY INDUCED ION CHANNELS

27

the kinetics and not the conductance of GABA-activated ion channels, but these effects have not been studied in sufficient detail to provide a mechanistic explanation for the convulsant-induced depression of the macroscopic responses. Modulation of pharmacologically activated ion channels by clinically important drugs may provide a means to identify synaptic currents. For example, one population of synaptic currents recorded in mouse spinal neurons decays exponentially with a time constant close to the average duration of an ion channel activated by GABA, as estimated by fluctuation analysis in the same cell (Barker and McBurney, 197913). The synaptic currents invert at the same potential as currents activated by GABA. Iontophoretically applied phenobarbital prolongs the time constant of decay of the physiologically elaborated current and increases the average duration of the GABA-induced ion channel event in the same cell. The drug produces little change in either the amplitude of the synaptic current or the conductance of the GABA-operated channel. Picrotoxin depresses the synaptic current without appreciably changing its time course and also depresses GABA responses recorded in the same cell (Barker et al., 1981b). The coincidence of inversion potentials, similarities in time constants, and parallel sensitivities of the physiological events and GABA-activated ion channels to phenobarbital and picrotoxin suggest that the synaptic currents may be mediated by GABA. If this is true, then one can estimate, given the average amplitude of a synaptic current and the conductance of an elementary event, that about 300 ion channels are open at the peak of the synaptic response. Similar results have been reported for quanta1 IPSCs mediated by GABA at invertebrate neuromuscular junctions (Dude1 et al., 1977). The pharmacology of GABA-activated ion channels in cultured spinal neurons should provide a useful reference for considering the physiology of GABA-mediated synaptic events in this system. These cultures are replete with terminal structures that stain for glutamic acid decarboxylase (the enzyme that decarboxylates glutamate to GABA) (Barker et al., 1981b), but it is not yet clear how functional these endings are. It would be of interest to compare the properties of C1- channels activated by GABA at subsynaptic membranes with those of channels opened by GABA at extrasynaptic sites. Differences in channel properties between synaptic and extrasynaptic sites may help to explain some of the wide variation in channel characteristics observed in results from cultured spinal neurons (Barker et al., 1981a). h. Single-Channel Currents Induced by Agonists in CulturedSpinal Neurons. The extracellular patch clamp method has been used to study the action of GABA, muscimol, and ( - )pentobarbital at the membrane of cultured mouse spinal neurons (Mathers et al., 1981). All three agonists induce rectangular jumps of inward membrane current of approximately 1.5-2.0 pA in amplitude at a membrane potential of - 80 mV (see Fig. 7). Analysis of records of this kind revealed that the closing of these agonist-induced channels is controlled by two kinetic

28

DAVID A. MATHERS AND JEPFERY L. BARKER

FIG.7. Single-channel currents induced by GABA and by the GABA analog muscimol in the membrane ofcultured mouse spinal cord cells. The GABA and muscimol records shown were obtained from two different neurons. Extracellular patch electrodes containing either 0.5 phf GABA 01-0.3PA4 muscimol were applied to the neuronal surface, electrode-membrane sealing resistances of about 50 M Q being obtained, A single KCI-filled intracellular microelectrode was used to polarize the cell membrane to -80 mV. Under these conditions, both agonists induce brief pulses of inward current of relatively constant amplitude but ofvariable durations. The bandwidth of the patch electrode circuit was DC-120 Hz.Temperature, 23OC. (M.B. Jackson, D.A. Mathers, J.L. Barker, and H. Lecar, unpublished observations.)

processes. The time constant of the slower of the two processes closely approximates that estimated from analysis of current fluctuations induced by these agonists when applied at moderate concentrations (Mathers and Barker, 1980a,b). The faster process has now been observed in current fluctuations induced by agonist concentrations lower than those used in previous studies (Mathers and Barker, 1981a). Further analysis is required to determine the nature of this fast component. C . VERTEBRATE AUTONOMIC GANGLION NEURONS Fluctuation analysis and relaxation methods have been used for the first time to investigate the nature of chemical transmission in neurons freshly dissected from the nervous system of adult vertebrates. The neurons employed in these studies (ganglion cells of the sympathetic and parasympathetic nervous system) are suitable for voltage-clamp studies because they are relatively accessible and lack extensive dendrites. 1. Nicotinic ACh Responses

Using both noise and relaxation methods, Ascher et al. (1979) analyzed the nicotinic response of rat submandibular ganglion cells (parasympathetic neurons) to ACh. Current noise spectra for ACh were initially reported to be of Lorentzian form, but further work revealed the presence of two kinetic components with time constants T~ = 35 msec and T~ = 7 msec at 2OoC (Rang, 1981). These time constants are both appreciably slower than values applicable

CHEMICALLY INDUCED ION CHANNELS

29

to ACh-operated channels in the vertebrate end plate (cf. Table I). As in the case of end plate ACh channels, 7, and .r, increased upon hyperpolarizing the cell membrane. Nerve-evoked EPSCs in these cells were found to decay with a biexponential time course. Furthermore, the time constants of this decay were in agreement with values of T~ and .r,calculated from ACh noise spectra. Interestingly, spontaneous miniature (min.) EPSCs decayed in a monoexponential manner with a time constant close to q. Kinetic analysis suggested that the two components seen in nerve-evoked EPSCs and in noise spectra represent two distinct classesofACh-operated channels. The absence of the slower process in min. EPSCs is possibly due to a spatial separation of these two channel types in the postsynaptic membrane (Rang, 1981). Assuming that the two populations of ACh-activated channels are of equal conductance, an estimate of? = 30 pS at 2OoCcan be made, a value close to that reported for end plate ACh channels (cf. Table I). The presence of two components in the ACh responses of ganglion cells contrasts with the simple Lorentzian behavior previously noted for ACh channels in the frog end plate and in Aplysia neurons (Gage, 1976; Ascher et a/., 1978a). However, complex kinetic behavior has also been reported in the case of AChsensitive channels in slow muscle fibers of the garter snake (Dionne, 1981b). In this preparation, kinetic analysis suggests that both components of the observed ACh noise spectrum reflect the complex gating of a “single’ ’ type of ion channel, in contrast to the conclusions reached by Rang in his study of ganglion cell responses (Rang, 1981). Analysis of the mode of action of several ganglionic blocking drugs on ACh responses of the rat submandibular ganglion cells revealed striking parallels with analogous studies previously carried out on Aplysiu central neurons (Ascher et al., 1978b). The agents trimetaphan and rnecamylarnine appeared to exert their antagonistic effect mainly by blocking the access of ACh to its receptors. In contrast, the antagonism produced by tubocurare, decamethonium, and hexamethonium was, as described in the case ofAplysiu neurons, largely due to the ability of these agents to block ion channels previously opened by cholinomimetics. This result is surprising, as tubocurare, decamethonium, and hexamethonium have long been considered as classic competitive antagonists of ACh action at the ganglion cell membrane. The outcome of these experiments therefore illustrates both the improved resolution offered by the new biophysical approaches to pharmacological problems, and the predictive value of quantitative invertebrate studies to the understanding of vertebrate neurophysiology.

2. Muscarinic A C h Responses A decisive step forward in the understanding of chemical transmission in autonomic ganglia was taken with the discovery, in bullfrog sympathetic

30

DAVID A . MATHERS AND JEFFERY L. BARKER

neurons, of a voltage-sensitive K + current that is inactivated by muscarinic agonists (Brown and Adams, 1980). In contrast to other voltage-dependent K + conductances in these cells, this so-called M current does not show appreciable electrical inactivation and therefore contributes substantially to determining the resting membrane potential. Voltage jump relaxation studies showed that the kinetics of the M current channels were slow (time constant ofrelaxation 7M = 150 msec at 22OC). T~ was voltage dependent, becoming shorter at more negative potentials. Muscarine suppressed the M current without greatly modifying its voltage sensitivity or the absolute value of T ~ It . was concluded that muscarinic agents depolarize and decrease the conductance of ganglion cells largely by depressing the amplitude of the M current. Because the application of muscarine closely mimics electrical events seen during the “slow EPSP” in these cells (Kuba and Koketsu, 1978), it seems probable that neurally released ACh also has access to the M current channels. Chemical inactivation of the M current may also account for the “late, slow EPSP” that is seen in bullfrog sympathetic neurons (Kuba and Koketsu, 1978) and that is believed to reflect the action of a peptidergic transmitter closely resembling luteinizing hormone releasing factor (LHRF) (Jan etal., 1979). It was shown that exogenous LHRF, like muscarine, depressed the M current without changingits kinetics (Fig. 8) (Adams and Brown, 1980). However, unlike theeffect of muscarine, the depressant action of LHRF was insensitive to atropine, suggesting that LHRF does not affect the M current indirectly by releasing ACh. These studies have provided a n economical explanation for the “ S ~ O W ” and “late, slow EPSPs” seen in bullfrog sympathetic neurons in terms of chemical inactivation of a common voltage-dependent K conductance by two distinct transmitter substances, ACh and an LHRF-like peptide. Caution should be ex-

-

+

4 pM LHRF

-

I

0.6B sec

mV

FIG.8. Responses of a voltagedamped bullfrog ganglion cell to 4 pMluteinizing hormone release factor (LHRF). The upper and lower chart records run continuously. Upper trace, time in seconds (with periodic 100-fold acceleration); middle trace, membrane potential (holding potential, -30 mV; command potential, -60 mV); lower trace, current responses (inward current downward). L H R F induces an inward current response that is associated with a decrease in membrane conductance. (Modified from A d a m and Brown, 1980.)

CHEMICALLY INDUCED ION CHANNELS

31

ercised, however, in extrapolating these ideas to synaptic events seen in other ganglionic neurons, in view of the many inconsistencies in the literature concerning this field (see Kuba and Koketsu, 1978).

IV. Conclusion

It is evident from this article that application of the fluctuation analysis, voltage jump relaxation, and extracellular patch clamp methods has provided much detailed information about the elementary events that underlie the response of nerve cell membranes to agonists. In addition, it has become clear that concepts originally developed to account for ACh action on skeletal muscle fibers appear also to describe the effects of ACh and certain other putative transmitters at a broad variety of neuronal membranes. Thus, a number of transmitter substances appear to utilize ion permeable channels to induce a charge transfer across the postsynaptic membrane. In most cases studied to date, putative transmitters act to open ion channels in the cell membrane. However, the chemical inactivation by ACh of M current channels in bullfrog sympathetic neurons provides an interesting exception to this trend. In addition, it has become evident that amino acid transmitters can also close ionic channels, as in the case of the GABA-sensitive K conductance reported in crayfish muscle fibers (Dude1 and Finger, 1980). It should be emphasized that, in the context of the functional organization of the vertebrate CNS, current knowledge of transmitter actions remains fragmentary. For example, at present, nothing is known about the elementary events that underlie the effects of glutamate and aspartate on CNS neurons, yet these amino acids are leading candidates for the role of excitatory transmitters in the mammalian brain and spinal cord (Nistri and Constanti, 1979). In addition, the actions of several other important classes of presumed transmitters, such as catecholamines, biogenic amines, peptides, purines, and pyrimidines have not yet been studied at the elementary level in any preparation. Do these substances also utilize ion channel mechanisms, and if so, how do the electrical properties of these channels compare with data obtained using other endogenous ligands? Is there a seeming randomness to the properties of channels activated by different substances, or will a pattern emerge, revealing a blueprint for elementary intercellular signals? Considerable technical problems exist in the application of the new biophysical methods to the study of transmitter actions on neurons in the intact vertebrate CNS. In the case of fluctuation analysis and voltage jump relaxations, control of membrane voltage at higher frequencies may be difficult to achieve in nerve cells with complex geometries. In addition, the membrane cur+

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DAVID A . MATHERS AND JEFFERY L. BARKER

rents recorded may reflect the summed activity of more than one receptor type, as application of an agonist cannot be localized easily to discrete parts of the neuronal surface. Interpretation of such complex signals in terms of kinetic models of agonist action is likely to prove difficult. The extracellular patch clamp is inherently less sensitive to the difficulties raised in the preceding paragraph. The technique does, however, require the presence of naked neuronal membranes in order to achieve a high electrode-cell sealing resistance. Unfortunately, most central neurons are at least partially invested by nonneural elements. It seems probable, then, that all three biophysical methods will be required in order to probe the action of putative transmitters on neurons in the intact vertebrate CNS. The most promising type of preparation for this venture would appear to be the brain and spinal cord slices already widely used in the electrophysiological investigation of C N S function. Within a few years, the elementary events induced by some transmitters in mammalian CNS neurons will become amenable to study without the need for cell dissociation and culture methods. This achievement will realize one of the long-standing goals of cellular neurobiology . REFERENCES Adams, P.R. (1975). Br. /. Pharmacol. 5 3 , 308-310. Adams, P.R. (1976). /. Physiol. (London) 260, 531-552. Adams, P.R. (1977). J. Physiol. (London) 268, 291-318. Adams, P.R., and Brown, D.A. (1980). Br. /. Pharmacol. 68, 353-355. Alger, B.E., and Nicoll, R.A. (1979). Nature(London)281, 315-317. Anderson, C.R., and Stevens, C.F. (1973). 1.Physiol. (London)235,655-691. Anderson, C.R., Cull-Candy, S.G., and Miledi, R . (1978). 1.Physiol. (London) 282, 219-242. Ascher, P., Marty, A,, and Neild, T.O. (1978a). /. Physiol. (London) 278, 177-206. Ascher, P., Marty, A , , and Neild, T.O. (1978b). J. Physiol. (London) 278, 207-235. Ascher, P. Large, W.A., and Rang, H.P. (1979). 1.Physiol. (London) 295, 139-170. Barker, J.L. (1975). Brain Rex 9 2 , 3 5 - 5 5 . Barker, J.L., and McBurney, R.N. (1979a). Nature(London) 277, 234-236. Barker, J.L., and McBurney, R.N. (1979b). R o c . R. SOL.London, Ser. B 2 0 6 , 319-327. Barker, J . L . , and Mathers, D.A. (1981). Science212,358-361. Barker, J.L., and Ransom, B.R. (1978a). /. Physiol. (London) 280, 331-354. Barker, J.L., and Ransom, B.R. (1978b). J. Physiol. (London), 280, 355-372. Barker, J.L., and Study, R.E. (1981). SOL.Neurosci. Abstr. 7 , 124. Barker, J.L., Nicoll, R.A., and Padjen, A. (1975). /. Physiol. (London) 245,521-536. Barker. J.L., McBurney, R.N., Mathers, D.A., and Vaughn, W. (1980). J Physiol. (London) 308, 18P. Barker, J.L., McBurney, R.N., and MacDonald, J.F. (1981a). J Physiol. (London)(in press). Barker, J.L., MacDonald, J.F., Mathers, D.A., McBurney, R.N., and Oertel, W. (1981b). In ‘‘Amino Acid Neurotransmitters” (F.V. DeFeudis and P. Mandel, eds.), pp. 281-293 Raven, New York.

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