Journal of Physiology (1996), 492.1, pp. 155-162

Spontaneous and uniquantal-evokedendplate currents in normal frogs are indistinguishable

William Van der Kloot

Department of Physiology and Biophysics, Health Science Center,State University of NewYork, Stony Brook, NY 11794-8661, USA

1. A recent paper concludes that the shapes of spontaneous and uniquantal-evoked signals aredifferent. The signals were recorded with extracellular electrodes, often in the presence ofneostigmine. Differences were reported between the voltage-time integrals, and betweenthe decay times of spontaneous and evoked signals.

2. These results disagree with earlier studies using the two-electrode voltage clamp technique.

3. We recorded miniature-endplate currents (MEPCs) and uniquantal-endplate currents(u-EPCs) in a low_Mg2+-Ca2+ solution, sometimes with neostigmine present. Evokedquantal outputs were estimated by the method of failures, so we could reject theappropriate number of the largest evoked releases. The twenty-nine experiments showedthat there were no consistent differences between the current-time integrals or half-decaytimes (t%), regardless of whether or not neostigmine was present.

4. When recording simultaneously with intracellular and extracellular electrodes, on averageabout 25% of the miniatures were seen in both recordings. On average, 63% of the end-plate potentials were also seen in both recordings. Extracellular recording may not give theprecise localization generally assumed.

5. We again conclude that quanta released by nerve stimulation and spontaneously areindistinguishable at normal frog neuromuscular junctions.

Cherki-Vakil, Ginsburg & Meiri (1995) presented evidencethat spontaneous and uniquantal-evoked releases, recordedwith extracellular electrodes, produce electrical responsesof somewhat different shapes at the frog neuromuscularjunction. We will call such signals, externals. They find thatthe evoked externals have larger voltage-time integralsthan the spontaneous externals. This contradicts a series ofcomparisons at the frog and mouse neuromuscular junctions,using intracellular recordings to measure the timeintegrals, of which they were unaware (Cohen & Van derKloot, 1983; Van der Kloot & Cohen, 1984; Van der Kloot,1987, 1991; Yu & Van der Kloot, 1991). Some novelfeatures in the experiments of Cherki-Vakil et al. (1995)persuaded us to do further measurements. Many of theirexperiments were done in the presence of the anti-cholinesterase neostigmine. They plotted decay times as afunction of amplitudes and fitted the points with regressionlines. The slopes of their regression lines are higher forspontaneous than for evoked externals.

The choice of extracellular recording by Cherki-Vakil et al.(1995) is questionable. When a current flows in through theendplate membrane the extracellular electrode detects apotential change produced by the current flow through theextracellular resistance. The amplitude of the externalsdepends on the magnitude of the current flow and on the

pathway through which the current flows between the tipof the electrode and site of inward current flow. As aconsequence, the amplitudes of the externals usually gradefrom a maximum down to signals so small that they areobscured by the instrumental noise. Examples of theamplitude distributions of externals are shown by delCastillo & Katz (1956; their Fig. 5). For this reasonexternals are quite unsuitable for the quantitativeexamination of the amplitudes of endplate currents.

The time course of the externals should follow faithfullythe current flow through the endplate channels. Therefore,they are suitable for measuring the rise and decay ofspontaneous miniature-endplate currents (MEPCs) and alsoof uniquantal-endplate currents (u-EPCs). However, whenrecording with an extracellular electrode how is itdetermined that a signal following nerve stimulation isgenerated by the release of a single quantum and that it isnot contaminated by the addition of smaller currentsflowing through loci on the endplate further from theelectrode tip? The fraction of the events seen by an intra-cellular electrode that is also recorded with an extracellularelectrode range from -1 to 50% (Cohen, Van der Kloot &Barton, 1981). Consequently with the extracellular electrodethere may often have been signals that were the sum ofseveral evoked quanta. In some experiments Cherki-Vakil

4565 155

1W Van der Kloot

et al. (1995) placed no Ca2+ in the extracellular solution. TheCa2+ for evoked release was provided by diffusion from theextracellular pipette, which contained 18 mm Ca2+. Thiswould limit the range of evoked releases but surely does noteliminate the possibility of undetected summations; itmight even increase this possibility. Therefore, we believethat externals are also unsuitable for measuring the timecourses of uniquantal-minature endplate currents(u-MEPCs).

These problems can be avoided by measuring with the two-electrode voltage clamp technique. The clamp detects allevents at the endplate. Quantal output can be reduced byworking in a low-Mg2+-Ca2+ solution. Yet even whenquantal output is low, some of the nerve stimuli release twoor more quanta, which may superimpose, so that theycannot be distinguished from a uniquantal release. Thisproblem can be minimized by using the method of failuresto estimate the mean quantal release, mo (del Castillo &Katz, 1954). Once mo is known, by Poisson's equation wecan estimate the predicted number of uniquantal releases,n,. We sort the EPCs in order of increasing size and selectthe n1 smallest fEPCs. These pruned data sets are the bestestimate that can be made for the distribution of u-EPCs.

The method just outlined was used to compare the sizes ofMEPCs and u-MEPCs at the frog and mouse neuro-muscular junctions (Van der Kloot & Cohen, 1984; Van derKloot, 1987; Yu & Van der Kloot, 1991). Size wasmeasured as the current-time integrals of the signals,fMEPC or fu-EPC. There was no difference in their sizes. Anumber of treatments enlarge quantal size by increasingthe quantity of acetylcholine in the packets. After quantalsize has been increased, the fMEPCs and fu-EPCs are stillindistinguishable. In the present paper we have alsomeasured the decay times of the signals and repeated themeasurements in the presence of neostigmine.

METHODSThe experiments were performed in the sartorius muscles of thefrog, Rana pipiens. The frogs were stored in the dark in runningwater at 4 °C and were killed by double pithing, in accordancewith the policies of the State University of New York at StonyBrook Animal Users Committee. Ringer solution contained (mM):120 NaCl, 2'0 KCI, 2'5 CaCl2 and 4-0 N-tris-(hydroxymethyl)methyl-2-aminoethanesulphonic acid (Tes)-NaOH, at pH 7 4. Forstudying evoked release with a low quantal output the solutionwas modified by adding 2-5 mm MgCl2 and reducing the CaCl2 to0 18-0 24 mm. The motor nerve was taken up into a suctionelectrode and stimulated using a Pulsar 6B stimulator (FrederickHaer & Co, Brunswick, ME, USA) and an A60 stimulus isolationunit (WPI, Sarasota, FL, USA). The stimulus duration was 80 #sand the intensity Mwas adjusted so that it was roughly three-times

threshold. The experiments were performed at room temperature,(18-22 °C).

Voltage clamping was done by the two-intracellular microelectrodemethod. The pipettes were slurry-bevelled to a resistance of1 5-3 MQ (Lederer, Spindler & Eisner, 1979). Ag-AgCl-coatedwires were used to connect to the electrodes and the solution in thetissue bath. Clamping was made with an Axoclamp 2A (AxonInstruments). The two electrodes were inserted into the same fibreat an endplate, with their tips -100 ,um apart. Then bothelectrodes were 'cleared', by oscillating the capacitive feedback,before the fibre was voltage clamped. The output of the voltageclamp was amplified further and filtered with an Axon CyberAmp320 amplifier, then viewed on an oscilloscope and fed through aDagan digital delay (Minneapolis, MN, USA) to a MetrabyteDAS16 A/D converter (Keithley, Taunton, MA, USA). The signalswere recorded over a bandwidth from 0-1 to 1000 Hz.

The holding potential of the voltage clamp was set at themembrane potential of the fibre when both electrodes wereinserted. Evoked release was initiated every 2 s by the computer,A/D conversion began and a pulse triggered the stimulator. Weexamined the signal and recorded whether or not there was anEPC. From the MEPCs and the u-EPCs we measured theamplitudes, the voltage-time integrals, JMEPC or fEPCs. Thenstarting from the peak signal, the computer determined the pointwhere the amplitude had fallen to half-peak. Then, starting fromthe end of the signal, the point where the amplitude had risen tohalf-peak, was also detected. The t% was the mean of the twoestimates. The method for measuring decay was tested by fittingan exponential to the signal from 90 to 10% of the peak amplitudeand using this equation to obtain alternative estimates of t%. Thepoints were fitted with a straight line to the logarithms of the datapoints with weighting of the data (Dempster, 1993). In fourexperiments, with 19 to 118 MEPCs, in no case was there asignificant difference between the two sets of measurements(Student's t test). The ratio of t; measured directly to t% estimatedfrom the exponential fit was 0X989 + 0 0059 (± S.E.M.). Althoughthe results were indistinguishable, the direct method waspreferred because in some instances, the fit to the exponential wasobviously poor.

The mean quantal outputs of the evoked releases, mo, wereestimated by counting the number of stimuli not followed by anEPC, no, and the total number of stimuli, N. Then:

mO = ln(N/no),(del Castillo & Katz, 1954). The number of uniquantal releases, n1,as predicted by Poisson's law, was calculated by:

ni = Nmo.Every data set was sorted in ascending order of fEPC, and thevalues associated with the smallest n, fEPCs were used to calculatethe measurements for u-EPCs.

Next, MEPCs were recorded from the same junction by passingthe signal directly from the CyberAmp to an Axon 2020 peakdetector. When the MEPC triggered the detector the pulse fromthe detector initiated the A/D conversion. Since the signal going tothe A/D converter first passed through the digital delay, the entire

J Physiol. 492.1156

Spontaneous and evoked quanta are alike

course of the MEPC was digitized. MEPCs were examined beforethe data were accepted, to eliminate overlaps.

Extracellular spontaneous and evoked potentials were recordedwith microelectrodes filled with 3 M KCl and with resistances of-2 MQ2 (Van der Kloot, 1995). The Ringer solution contained(mM): 0 4 CaCl2, 2 5 MgCl2 and 20 sucrose (to raise the rate ofspontaneous release). The recording bandwidth was from0-1-10 kHz. The complete time course of both signals wasrecorded by use of a CIO-DAS16/330 board and software fromComputer Boards (Mansfield, MA, USA), which can store a setnumber of points before a trigger.

There is considerable evidence that the sizes of synaptic potentialsdo not fit normal probability distribution functions (Van der Kloot,1987; Bekkers, Richerson & Stevens, 1990; reviewed by Van derKloot, 1991). Therefore, statistical tests were done using the non-parametric Kolmogorov-Smirnov goodness of fit test.

RESULTSDecay timesThe results of the twenty-nine experiments aresummarized in Table 1 and examples of the data are inFig. 1. The mean mo was 0A44, while the mean numbers ofu-EPCs and of MEPCs were 94 and 135, respectively. Someof the experiments were done in 0 3 nM neostigmine(10-6 mg mI-l), the extremely low concentration apparentlyused by Cherki-Vakil et al. (1995). Others were done in3/M neostigmine. Consider first the distribution of t; anexample is in Fig. 2A. In three of the data sets thedifferences between the t% of u-EPCs and the MEPCs werestatistically significant with P < 0 05. In one of theseinstances the u-EPCs were shorter than the MEPCs. In two

Table 1. Summary of evoked and spontaneous releases

mO Evoked Spont. Ju-EPC(pA s-')

0 49 100 100 6'10-46 102 105 12-80-38 71 120 9.90 35 101 120 14-30'30 85 203 8*01-17 44 III 10-41-17 109 100 5-80 45 101 151 7'60 09 80 100 4-10-08 25 80 6-20.19 100 151 6-10-23 103 101 4-61.05 150 101 19.00-29 100 151 8-40-21 61 61 6-5

In 0'3 nM neostigmine-70 0-16-60 0 40-75 0 44-75 0.91-80 0'24-80 0 35-90 0 39-85 0 50

In 3/uM neostigmine-60 0-24-60 0 39-70 0-89-75 0'28-50 0-36-50 04

68 100 12-680 252 7-7118 252 12-0100 209 10-788 102 12-583 83 15 081 150 9.3152 150 9-3

71 127 16-5108 152 16-2123 151 17-495 130 20-892 102 8*4

121 201 12-1

fMEPC(pA s-)

5.310*19.0

11.99.16-54.95.94.45*045.03-85.96-34*1

14-97*6

11*110-712-113-98'98*6

20-016-817-923-810.912 1

Prob fs t,u-EPC

0-160.990.0010.590.010-890.090.150.0010-200-480-0060.00010-230-008

00070.110'070-430590-060-22004

0040-360300-140-060'42

(ms)1.52-62-12 12-11-21*0

1.01.01-41P41P21.91-51'2

t4MEPC(ms)1X42X42-12-12-41.20.91P01'01.21*51-21 61P51-2

Prob &tj

0-970940X230-450X010X890X820X680X230090-940-490X00010.90-17

2'9 2-1 0-082-1 2-2 04112-7 2-4 0.0012-3 2-4 0-572'3 2-4 0-142-4 2-5 0.592-7 2-7 0 452-6 2-6 0-69

2-7 2-7 0 542X9 2-9 0-883.3 3.3 0-362-5 2-7 0 072-5 2-7 0492-1 2-3 0-13

When P < 0 05 the values are printed in bold; Vh, holding potential; prob, probability.

Vh(mV)-72-86-85-85-80-85-90-91-86-85-90-80-95-80-75

J Physiol. 492.1 157

Wv Van der Kloot

AIe.*

5 nA

5 MEPC5 ms

B

EPC

1 nA

5 ms

Figure 1. Examples of the recordingsA, an EPC and a MEPC in a preparation clamped at -80 mV. B, mean MEPCs and EPCs from apreparation clamped at -60 mV. The MEPC is the mean of -200 results. The EPC is the mean of80 results. Since mo was 0 4, about 19 of the EPCs contained more than 1 quantum.

examples the u-EPCs were longer than the MEPCs. In oneof these two the probability of the differences occurring bychance was very low, P < 10-4. This data set will bediscussed shortly and we will argue that it should bediscarded. Our overall conclusion is that the decay times areindistinguishable between u-EPCs and MEPCs, whether ornot neostigmine is present.

Current-time integralsAlso shown in Table 1 are the mean fu-EPCs and fMEPCs.An example of their distributions is shown in Fig. 2B. In

A

eight of the data sets the fu-EPCs and JMEPCs weresignificantly different. In four of these the fMEPCs werelarger than the fu-EPCs, while in the remaining four thereverse was true. The overall conclusion is that thecurrent-time integrals are indistinguishable between theMEPCs and the u-EPCs, regardless of the presence ofneostigmine.

In one example the differences were very large; thefu-EPCs were more than three times as large as thefMEPCs. This is the same example that was mentioned

B

oD o"0

' MEPC

1*0

0-8

._

-

0.D

a)._..

E0

0-6

0-4

0-2

0-02 3 4 5 6

ti (ms)0 5 10 15 20

fMEPC (pA s-1)

Figure 2. Cumulative plots of the distributions of t. and JMEPCs at a junction held at -50 mVin solution containing 3#M neostigmineA, t; of 201 MEPCs (mean, 2X3 ms) and 121 u-EPCs (mean, 2X1 ms). B, current-time integrals for thesame data sets. The means of both were 12 1 pA.

158 J. Physiol. 492.1

1*0

0-8

0-6

0-4

Cu.0

co02.

coCu

E0

0

MEPC,,

0-2

0-025 30

Spontaneous and evoked quanta are alike

above, in which the t, values of the u-EPCs were longerthan those of the MEPCs, with P < 10-4. We suspect thatin this preparation, conduction was sometimes blocked inthe motor nerve before the action potential reached theterminal. When the action potential was conductedthrough, a number of quanta were released in many of theEPCs. However, vhen the action potential was blocked, theEPC was zero. This could account for this data set. One wayto check the interpretation would have been to repeat themeasurements, stimulating with a pair of pulses separatedby an interval of 50-100 ms. The second stimulus of thepair often overcomes a conduction block (Barton & Cohen,1982). Unfortunately, in this example the electrodes cameout of the fibre before this test could be done. We believethat this data set should be disregarded.

Plots of t% as a function of fMEPCCherki-Vakil et al. (1995) plotted decay time as a functionof amplitude of each of the signals. They then calculated aregression line for the data. They found that the regressionline for plots of decay time as a function of spontaneousexternal amplitude had significantly positive slopes. Theslope for decay time as a function of external-evokedamplitude was low and often indistinguishable from zero.We did the same calculations on our data. In sixteen casesthe slope of the regression line for the MEPCs was greaterthan the slope for the u-EPCs. In the remaining thirteeninstances the slope for the u-EPCs was higher than that forthe MEPCs. An example of these plots is in Fig. 3. Here thefit of the lines to the points were statistically significant

A6

5

4

ti (ms) 3

2

1

0 _0

0

* 0

* 0

A., I *

2 4 6

u-EPC (nA)

Table 2. MEPPs accompanied by an external

Numberof MEPPs

105216307124144271

Numberof simultaneous externals

155330673478

(P < 0 05). This was the case in only twenty-eight of thefifty-eight examples. We conclude that there is noconsistent difference in the slopes.

Problems with the use of extracellular recordingThe usual assumption is that an extracellular electroderecords from a circumscribed area of the endplate. This isdifficult to test directly, but we can record simultaneouslywith intracellular and extracellular electrodes (Fig. 4A andB). As reported by a number of groups (references in Cohenet al. 1981), an appreciable percentage of the miniatureendplate potentials (MEPPs) are often recorded by bothelectrodes. In six experiments made with preparations usedin this study 26-0 + 6-3% MEPPs were accompanied by anextracellular signal (Table 2). In a terminal with 500 releasesites, we must either be detecting events generated byrelease from a number of sites, or release at the recordingsite must be high. This might be owing to local stimulationof spontaneous release by the presence of the extracellularelectrode. However, the positioning of the extracellular

B6

5

44 ~ ~ ~ ~ o ..

t (ms) 3 t * 0g.e ~ *1,

2~~~e'2 tVW..~~~4 *

0 2 4 6

MEPC (nA)

Figure 3. Plots of t, as a function of amplitudes in a preparation in 3 EM neostigmine clampedat -50 mVA, 121 u-EPCs. The slope is 0-31 (correlation coefficient, r2, 0 15). B, 201 MEPCs. The slope is 0'27

0,0-07).

159J Physiol.492.1

1W Van der Kloot

Table 3. Simultaneous EPPs and externals

EPPs with asimultaneous external MEPPs

Number of stimuli2009998

(%)

368867

(MO ± S.E.M)1-54 + 0132-03 + 0-263-49 + 057

electrode did not appear to increase MEPP frequency.Cohen et. at. (1981) showed that MEPP frequency could beunchanged when the extracellular electrode was put intoposition, withdrawn, and then repositioned once again.

We also examined evoked potentials with intracellular andextracellular electrodes (Table 3). On average, 63% of theendplate potentials (EPPs) were accompanied by an

external. From the data sets we also estimated quantaloutput, in, using the signals from the intracellular or theextracellular electrode (Table 3). The extracellularmeasurement is sometimes arbitrary, because the signalamplitudes fall off until they are obscured by background

A -. i A..

02 mV

10 ms

C

2 mV

5 ms

noise. We counted all the amplitudes that fell below an

arbitrary threshold as a failure (Fig. 4C and D). Threeexperiments showed that the mo estimated by theextracellular electrode was between 22 and 71% of thatestimated with the intracellular electrode (Table 3). Againthese results suggest that the localization achieved by theextracellular electrode was by no means as precise as

commonly assumed.

In three experiments we measured the t% of EPPs andMEPPs recorded with an extracellular electrode. The t% ofthe spontaneous signals were 70 7 + 6X7% of the X, of theevoked signals.

B

0.2 mV

10 ms

D

Figure 4. Examples of simultaneous measurements with an intracellular and an extracellularelectrodeA and B, MEPPs in the lower traces and externals in the upper traces. The MEPPs are small and noisybecause the fibres had low resting potentials. The traces were selected to illustrate a high level ofcoincidence. C and D, EPPs in the upper traces and externals in the lower. The external in panel C was

judged to show an evoked response, while that in panel D was taken as a failure.

Externals(MO ± S.E.M.)034 + 00451P46+0-181-06+0-139

160 J Phy8iol.492.1

Spontaneous and evoked quanta are alike

DISCUSSIONThe idea that spontaneous and evoked quanta are the samewas an assumption underlying the experiments thatestablished the concept that the endplate potentials are thesum of individual MEPPs (del Castillo & Katz, 1954; Boyd& Martin, 1956). Elmqvist & Quastel (1965) showed that inhuman muscle the amplitudes of spontaneous and evokedquanta fell in tandem as the quantal size was diminished bythe action of hemicholinium-3. Spontaneous and uniquantalpostjunctional currents measured with the cell-attachedpatch in the hippocampus also have indistinguishableamplitudes (Raastad, Storm & Andersen, 1992). However,if there are relatively small differences, like those proposedby Cherki-Vakil et al. (1995), they would not have seriouslyinterfered with the quantal analysis and might not beapparent in amplitude measurements.

Spontaneous and uniquantal-evoked releases werecompared in detail by Van der Kloot & Cohen (1984), usingmethods like these in the present paper. Recordings weredone at temperatures ranging from 5 to 15°C and atholding potentials ranging from -97 to -140 mV. In onlytwo of the seventeen examples did spontaneous and evokedsignals differ significantly, so it was concluded that theywere the same. Additional measurements were done onpreparations treated to increase quantal size (Van derKloot, 1987) and at the mouse neuromuscular junction (Yu& Van der Kloot, 1991). Again there were no consistentdifferences between spontaneous and evoked releases. Weperformed the present experiments because of the evidencepresented that there are differences in the decay times ofspontaneous and evoked quantal currents and to check onthe possibility that there are differences in the slopes ofplots of decay times as a function of amplitude. The presentexperiments agree with our earlier results that there is noindication of consistent differences between the evoked andspontaneous quanta. As argued in the introduction, webelieve that the contrary results of Cherki-Vakil et al.(1995) can be accounted for by their use of extracellularrecording. It is widely assumed that extracellular recordingdetects only events at highly circumscribed regions of theendplate. As discussed by Van der Kloot & Molgo (1994),this assumption is not well supported by experiments. Thefact that so many of the intracellularly recorded MEPPs areaccompanied by a signal detected by the extracellularelectrode suggests that localization may be much poorerthan assumed. The large fraction of evoked outputs, alsodetected by the extracellular electrode, also argues againstprecise localization. The circuit for extracellular recordingat the endplate may be more complicated than commonlydrawn (Van der Kloot & Molgo, 1994).

Glavinovic (1987) concluded that during synaptic depressionthe evoked quanta become smaller than the MEPCs. The

preparation was stimulated tetanically to producedepression. The solution was changed to one with a low[Ca2P]. Recording was then begun to estimate mo. In a trainof 437 stimuli two zeros were observed. It is by no meansclear that release was stationary during the measurement,so the estimate of mo may be too high. The size of theevoked quanta was calculated by dividing the mean EPCamplitude by min. If mo is too high then the estimate of theevoked quantal output will be too low. Another widely citedreason for thinking that spontaneous and evoked quantacome from different populations comes from studies of therate of incorporation of the false transmitter, acetyl-monoethylcholine, into quanta in the rat (Large & Rang,1978). The presence of the false transmitter can be detectedbecause it opens endplate channels for a shorter meanduration. Large & Rang (1978) exposed preparations to theprecursor monoethylcholine. They then compared EPPsrecorded in curare and MEPCs, concluding that the falsetransmitter is incorporated more rapidly into evokedquanta. We have restudied this question in the frog byrecording MEPCs and EPCs, and conclude that thespontaneous and evoked quanta probably incorporate falsetransmitter at the same rate (Van der Kloot, Naves &Balezina, 1995).

After certain unusual treatments, spontaneous and evokedquanta are clearly different (reviewed by Molg6, Pecot-Dechavassine & Angaut-Petit, 1989; Van der Kloot, 1991).This is shown after poisoning with botulinum toxin type Aor treatment with 4-aminoquinoline (Colmeus, Gomez,Molg6 & Thesleff, 1982; Molg6 & Thesleff, 1982; Thesleff,Molg6 & Lundh, 1983). Here, the number of spontaneouslyreleased quanta that are slowly rising are often large, andthey are not usually released by nerve stimulation. Manyquestions remain to be resolved about such quanta, but itmay be counterproductive to include them when consideringthe great majority of the quanta released from untreatednerve terminals.

To conclude, at present there appears to be no solid evidencethat under normal conditions the spontaneously releasedquanta generate signals that can be distinguished fromthose generated by evoked quanta. Over the years therehave been many speculations about the physiological role ofspontaneous release. Such speculations may be misleading.When the rate of spontaneous release is compared with themaximum rate of evoked release, the ratio is similar to thatmeasured for chemical reactions when they are spontaneousand enzymatically catalysed (Van der Kloot & Molgo,1994). In other words, spontaneous releases may simply bea necessary consequence of the thermodynamics of therelease process. The release of evoked and spontaneousquanta from a single pool is implicit in this interpretation.

J. Phy8iol.492.1 161

162 W. Van der Kloot

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AcknowledgementsThis work was supported by grant number 10320 from theNational Institute of Neurological Disorders and Stroke. I thankJudy Samarel for assistance.

Received 2 May 1995; accepted 3 November 1995.

Neurobiology 36, 93-130.