Proc. Nati. Acad. Sci. USAVol. 83, pp. 8410-8414, November 1986Neurobiology

Action-potential duration and the modulation of transmitter releasefrom the sensory neurons of Aplysia in presynaptic facilitation andbehavioral sensitization

(K+-channel modulation/serotonin/learning/memory)

BINYAMIN HOCHNER*, MARC KLEIN, SAMUEL SCHACHER, AND ERIC R. KANDELHoward Hughes Medical Institute, Center for Neurobiology and Behavior, Columbia University, College of Physicians and Surgeons, and The New York StatePsychiatric Institute, New York, NY 10032

Contributed by Eric R. Kandel, July 16, 1986

ABSTRACT Presynaptic facilitation of transmitter releasefrom Aplysia sensory neurons is an important contributor tobehavioral sensitization of the gill and siphon withdrawalreflex. The enhanced release is accompanied by reduction of theserotonin-sensitive S current in the sensory neurons and aconsequent increase in duration of the presynaptic actionpotential (ranging from 10% to 30%). We find that changes ofsimilar magnitude in the duration of depolarizing voltage-clamp steps in sensory neurons in intact abdominal gangliayield increases in synaptic potentials of 45-120%. In dissoci-ated cell culture, these changes lead to increases of 25-60% inthe synaptic potential. Prolongation of presynaptic depolariza-tion using voltage clamp or prolongation of the duration of theaction potential by K+-channel blockers leads to prolongationof the time-to-peak of the synaptic potentials; similar changesin time-to-peak occur during presynaptic facilitation. Thetime-to-peak is not changed by homosynaptic depression or bychanging the Ca2' concentration, procedures that alter releasewithout changing the duration of the action potential. Prevent-ing the spike from broadening by voltage clamping thepresynaptic neuron substantially reduces or blocks the facili-tation. These results suggest that broadening of the actionpotential during facilitation is a causal factor in the enhance-ment of transmitter release.

Short-term sensitization of the gill and siphon withdrawalreflex of Aplysia leads to facilitation of transmitter releasefrom the presynaptic terminals of the sensory neurons ontotheir various target cells, the interneurons and the motorneurons (1). In earlier work, we described two changes thataccompany presynaptic facilitation in sensory neurons: (i) adecrease in the S current, a serotonin (5-HT)-sensitive K+current (2-5); and (ii) an increase in Ca2+ transients measuredwith the Ca2l indicator arsenazo III (6). We proposed that thedecrease in K+ conductance augments transmitter release byprolonging the action potential and thereby increasing Ca2+influx into the terminals; in addition, the decrease in K+conductance also increases the excitability of the neurons (2,3, 7-9). Although the function of the alteration in Ca2`handling is not known, Boyle et al. (6) suggested that it mightact synergistically with the spike broadening to enhancetransmitter release.

This is the first of two related papers designed to analyzethe relative contributions to presynaptic facilitation of spikebroadening and other potential contributing processes, suchas the altered handling of Ca2'. In this paper, we attempt todetermine the degree to which spike broadening contributesto presynaptic facilitation. We find that broadening producedby facilitating stimuli has an important effect on transmitter

release, and could account for much of the facilitationproduced by sensitizing stimuli.

Broadening is not, however, the only determinant offacilitation. In the subsequent paper (10), we show that anadditional set of facilitatory processes becomes dominantwhen transmitter release is depressed, as occurs after therepeated activation of the sensory neurons that accompaniesbehavioral habituation.

METHODSAbdominal ganglia were removed from Aplysia californicaweighing 100-250 g (Pacific Biomarine, Venice, CA; Sea LifeSupply, Sand City, CA). The ganglia were immersed for 45sec in 0.5% glutaraldehyde [to prevent sheath contractions(11)] in artificial sea water [ASW; 460 mM NaCl/10 mMKCl/11 mM CaCl2/55 mM MgCl2/10 mM Tris or Hepes(Sigma), pH 7.6], rinsed in ASW, desheathed, and pinned outon Sylgard. In the experiments in culture, abdominal orpleural sensory neurons and followers were dissociated,plated, and grown by the method of Schacher and Proshan-sky (12). To prevent spiking in followers, they were heldhyperpolarized. Voltage clamping was done with a Dagan8500 two-microelectrode voltage clamp; microelectrodeswere filled with 2.5 M KC1. Low-ionic-strength solutioncontained 50 mM NaCl, 10 mM KCl, 33 mM CaCl2, 25 mMtetraethylammonium chloride (Et4NCl, Kodak), 10 mM Tris,and 0.5 M sucrose (Sigma). For high-divalent-cation seawa-ter, ASW was modified to contain 260 mM NaCl, 60 mMCaCl2, and 140 mM MgCl2. Other compounds used were5-HT-creatinine sulfate complex (Sigma) and 3,4-diamino-pyridine (3,4-DAP, Sigma). All experiments were done atroom temperature.The shapes of (excitatory) postsynaptic potentials [(E)PSPs]

were analyzed with a Prowler laboratory computer (Norland,Ft. Atkinson, WI). In some experiments EPSPs were aver-aged with a signal averager. To calculate the relative contri-butions to the changes in amplitude of changes in time-to-peak (T) of the EPSP and in its rate of rise (R), we used thefollowing formula:

% contribution of T =

log POS x 100Tpre

log TPOSt + log RPOStTpre Rpre

where Xpre is the value ofX before the procedure that alteredEPSP amplitude and Xpost is the value afterwards. The

Abbreviations: 5-HT, serotonin; Et4N', tetraethylammonium; 3,4-DAP, 3,4-diaminopyridine; (E)PSP, (excitatory) postsynaptic poten-tial; ASW, artificial sea water.*Present address: Department of Neurobiology, Life Sciences In-stitute, Hebrew University, Jerusalem, Israel.

8410

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. NatL. Acad. Sci. USA 83 (1986) 8411

relative contribution of R was taken as 100 minus thepercentage contribution of T. This formula was based on thesimplifying assumption that the early part of the rising phaseof the EPSP can be approximated by a straight line; the peakamplitude of the EPSP would then be equal to the product ofR (the slope of the rising phase) and T. The ratios of the postvalues to the pre values were converted to logarithms so thatthe contributions of T and R could be expressed additivelyrather than as a product. The true amplitude is less than theproduct ofR and T because the later part of the rising phasehas a smaller slope than the earlier phase. As a result, therelative contributions of changes in T and R to alterations inamplitude should be considered as approximations only.

RESULTS

Application of 5-HT, stimulation of the facilitating pathwayfrom the head or tail, and injection of cAMP or of thecAMP-dependent protein kinase all produce an increase inthe duration of action potentials in sensory neurons (2, 3, 7,13, 14). In the absence of drugs that depress other K+currents, this increase is 10% in the cell bodies of intactabdominal ganglion sensory neurons (2), 15% in the cellbodies of abdominal ganglion sensory neurons in dissociatedcell culture (15), and 25-30% in the cell bodies or growthcones of the pleural ganglion sensory neurons in vivo or inculture (14). Since these action potentials terminate substan-tially before the peak ofthe Ca2l current is reached, and sincetransmitter release is thought to vary exponentially with theCa2l current (16), small changes in duration could have largeeffects on transmitter release. To determine if the amount ofbroadening actually observed contributes importantly topresynaptic facilitation, we performed experiments on ab-dominal sensory neurons, both in intact ganglia and inculture, and on pleural sensory neurons in culture.There Is a Steep Relationship Between the Duration of the

Presynaptic Command Pulse and Transmitter Release inUndepressed Synapses. If prolongation of the duration of theaction potential contributes significantly to presynaptic fa-cilitation, and if the change in action potentials recorded inthe cell body is indicative ofwhat happens at the presynapticterminals (see ref. 14), then dependence oftransmitter releaseon the duration of the action potential should be quite steep

in the range of 2-3 msec, the normal duration of the sensoryneuron spike (see also ref. 17).We first examined the dependence of release on the

duration ofpresynaptic depolarization under voltage clamp inintact abdominal ganglia. We found that we obtained the bestvoltage-clamp control over release when we used solutionswith a low ionic strength and added Et4N'. Under theseconditions, transmitter release was graded as the depolar-izing command was increased in amplitude. Small increasesin pulse duration in the range of normal action potentials(about 2.0-3.0 msec) gave graded and relatively large in-creases in the amount of transmitter liberated from theterminals (Fig. 1A). In the average of three experimentsstarting from initial durations of 2.5-3.0 msec, increases induration of 10, 20, and 30%o produced increases in transmitterrelease of 45, 84, and 121%, respectively. Thus, the changesin duration produced by sensitizing stimuli in normal seawater could increase transmitter release substantially.To obtain better control of transmitter release in more

normal bathing solutions, we carried out a series of experi-ments in which we examined the dependence of transmitterrelease on duration of presynaptic depolarization in dissoci-ated cell cultures. By plating sensory neurons close to motorcells, we could shorten the distance from the cell body to theterminals and achieve better voltage control of the terminalswithout modifying the composition of the normal bathingsolution.

In dissociated cell cultures from pleural sensory cells, nowin the presence of normal bathing solution, release againproved quite sensitive to changes in duration (Fig. iB). In theaverage of three experiments starting from a step duration of2 msec, increases in duration of 10, 20, and 30% enhancedtransmitter release by 25, 46, and 62%, respectively. If theincreases in duration that were seen in pleural cell bodies inculture (25-30%) were similar to those occurring in theterminals, as work on growth cones suggests (14), then theincreases in action-potential duration produced by connec-tive stimulation or 5-HT would contribute significantly topresynaptic facilitation.

Alterations in Duration of the Action Potential ProducePredictable Changes in the Shape of the EPSP. Our conclu-sions about what happens in the presynaptic terminals rest inlarge part on indirect arguments based on observations in thecell body. To gain more direct access to events at the

A

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B

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XA 1

02.5 5 7.5 10Step duration, msec

C

J 2.5 mV0.5 sec

20Post 10 mV

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Pre +12mVI

E 15

1100 nAl

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Vm

-50 mV J V- m

5 msec

10

1 2 3 4 5 6 7 8 9 10Step duration, msec

FIG. 1. Dependence of transmitter release on presynaptic step duration. (A) A sensory neuron in an intact abdominal ganglion wasvoltage-clamped at -50 mV in low-ionic-strength bathing solution, and the voltage was stepped to +40 mV. PSPs of different amplitudes wereelicited as the step was changed in duration. The top part of the figure shows samples of the data from which this plot was constructed. (B Left)Voltage-clamp steps from -50 mV to +12 mV in a sensory neuron (Pre) in cell culture were increased in duration and gave rise to progressivelylarger EPSPs in a follower neuron (Post). (B Right) Plot of the dependence of transmitter release on presynaptic step duration (same cell as inB Left). Im. membrane current; Vm, membrane voltage. Bathing medium was normal ASW. The curves in this and all subsequent figures weredrawn by eye.

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8412 Neurobiology: Hochner et al.

terminals, we tried to infer some of the properties of thepresynaptic depolarization from observation of the shape ofthe postsynaptic response.We noted, as have Llinas et al. (18), that when a neuron is

voltage-clamped and the depolarizing command is prolongedwell past the release threshold, the time-to-peak of thepostsynaptic potential is progressively increased (Fig. 1BLeft and 2A). Similar changes in time-to-peak occurred whenthe spike was broadened as a result of application of theK+-channel blockers Et4N' or 3,4-DAP. Fig. 2B Rightillustrates the broadening of the action potential and theincrease and prolongation of transmitter release caused by3,4-DAP. By contrast, when synaptic transmission wasaltered without changing the duration of the spike, as bybathing in medium containing different Ca2l concentrationsor during homosynaptic depression, the shape ofthe PSP wasunchanged (Figs. 2B Left, 3A Top and Middle, and 3B).The absence of a shape change With altered Ca2' and with

homosynaptic depression implies that the absolute level oftransmitter release can be altered substantially without anynecessary concomitant change in the shape of the PSP. Sincethe somatic action potential broadens during facilitation,finding that the facilitated EPSP is also prolonged wouldsupport the idea that prolongation of the action potential alsooccurs in the presynaptic terminals and that this broadeningis causally related to presynaptic facilitation..

Presynaptic Facilitation Is Accompanied by Changes in theShape of the PSP. We therefore examined synaptic potentialsin intact ganglia before and after stimulation of a facilitatorypathway. We found a marked prolongation ofthe rising phaseof the EPSP (Fig. 3A Top and Middle). The average increasein time-to-peak in normal seawater was 18% ± 7 (n = 6) and43% ± 18 (n = 6) in high-divalent-cation medium. Althoughthe time-to-peak changed significantly, the later decay phasewas not changed, making it unlikely that a change in passiveproperties of the postsynaptic membrane is responsible forthe altered configuration of the synaptic potentials (Fig. 3ABottom). Direct measurement of membrane resistance inother experiments also revealed no change. Thus, the pro-longation of the EPSP that accompanies presynaptic facili-tation is consistent with a prolongation in transmitter releasefrom the presynaptic site. As pointed out above, there is littleor no change in the EPSP configuration during homosynapticdepression (Fig. 3A Top and Middle and 3B). This suggeststhat depression and facilitation diverge in mechanism at somepoint.

Prevention of Spike Broadening Reduces Presynaptic Facil-itation. The experiments we have considered so far areconsistent with a mechanism for presynaptic facilitation that

involves broadening of the action potential as one importantcomponent. However, our data do not allow us to excludecontributions of other components to this facilitation. Thisquestion is particularly relevant because earlier we had founda second, independent effect associated with presynapticfacilitation: the Ca2l concentration transient caused by adepolarizing step is directly modulated by 5-HT (6). Thissuggests that the cell's handling of Ca2l is altered and mightbe involved in facilitation of transmitter release. Since Ca2+handling may involve intracellular processes, it is possiblethat changes in such a process might not be detected byexamining the membrane currents alone. Nevertheless, if thechange in K+ conductance is important in facilitation, acausal connection between the K+ conductance decreasesand facilitation should be demonstrable.The only obvious way that a K+ conductance decrease

could be translated into increased transmitter release is bymeans of an effect on the membrane potential or the actionpotential. This effect might be a change in the steady-statepotential, enhancement of a passively conducted depolariza-tion, or a change in the number or configuration of actionpotentials (9). Preventing potential changes from occurringby means ofan adequate voltage clamp of the sensory neuronterminals should then reduce facilitation, despite the fact thatthe K+ conductance could still be modulated.We were not able to achieve adequate voltage control in the

intact ganglion to allow us to perform this experiment in amanher that would be interpretable. However, in culture, wewere in some cases able to elicit graded transmitter releasefrom a presynaptic neuron under conditions of good voltagecontrol and to examine the effects of 5-HT. Fig. 4A Leftillustrates the characteristic duration-release curve undervoltage clamp. Later in the experiment, 5-HT was applied,and a curve was again generated. The curve obtained after theapplication of 5-HT was superimposable on the controlcurve, except for the longest pulse duration (10 msec). Thissuperimposability suggests that when the synaptic potentialis not depressed, the input-output curve is not significantlyaffected by 5-HT except at the longest duration. Under thesecircumstances, processes other than spike broadening appearto contribute little to facilitation.

Fig. 4A Right and B illustrate a more limited input-outputcurve in an experiment in which voltage-clamp and current-clamp experiments could be compared in the same cell. Aninput-output curve was first generated under voltage clamp.As before, adding 5-HT did not significantly affect theamplitude of the EPSP at any duration except perhaps thelongest. The experiment was then repeated on the same cellthe next day under current clamp (Fig. 4C). 5-HT now

A

Post

B

400tA

+12 mV -l

Pre I-m-50 mV ?, I

5 msec

10 mV

|2 mV

I50 mV

10 msec

FIG. 2. Effect of duration of the presynaptic depolarization on the shape of the EPSP. The two parameters that determine the amplitudeof an EPSP are its rate-of-rise (or initial slope of its rising phase) and its time-to-peak amplitude (see Fig. 3B Inset). Increasing either of thesewill increase EPSP amplitude. (A) Once the threshold for the EPSP has been exceeded, increasing the duration of presynaptic voltage-clampdepolarizations leads to an increase in the time-to-peak of the EPSP (A) without affecting the initial rate-of-rise, or slope. (B Left) Duringhomosynaptic depression, there is no change in the presynaptic action potential (bottom trace) and the time-to-peak of the EPSP remainsconstant, whereas the rate-of-rise decreases (top traces). (B Right) Broadening the presynaptic action potential with the K+-channel blocker3,4-DAP (0.1 mM) (bottom traces) leads to an increase in EPSP time-to-peak (top traces, A) without changing the rate-of-rise.

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Proc. Natl. Acad. Sci. USA 83 (1986) 8413

A

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PSP amplitude

Rate-of-rise

Time-to-peakHeterosynapti

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0 500 >

E 40[ 0 30o0~~~~~l

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C

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10 msec

Current clamp

5-HT4

Post

Pre

:C

3,4-DAP

0%

FIG. 3. Changes in the shape of the EPSP accompanyingpresynaptic facilitation. (A Top) Superimposition of the first EPSP ina homosynaptic depression series (largest EPSP) and two EPSPslater on. There is relatively little change in time-to-peak duringhomosynaptic depression (A). (A Middle) The smallest EPSP is oneofthe depressed EPSPs fromA Top, while the two larger EPSPs wereelicited after presynaptic facilitation induced by connective stimu-lation. In the facilitated EPSPs, the time-to-peak is substantiallyincreased. Bathing medium contained high amounts of divalentcations. (A Bottom) In another sensory neuron, EPSPs from cellsbathing in normal ASW were elicited before and after connectivestimulation, and the decay phases were compared. Twenty EPSPsbefore and 20 after connective stimulation were averaged, and thetwo averages were normalized with respect to amplitude. Theaverage of the facilitated group was somewhat larger and longer thanthat of the control group, yet shifting this record to the left shows thatthe decay phases are essentially superimposable. This suggests thatthe change in EPSP duration cannot be accounted for by a change inpassive postsynaptic membrane properties. (B) These histogramscompare the relative contributions of rate-of-rise and time-to-peakchanges in EPSP amplitude under several conditions: (i) change inextracellular Cae' concentration; (ii) homosynaptic depression; (iil)heterosynaptic facilitation caused by connective stimulation; and (iv)

blockade ofK+ channels with 3,4-DAP (0.1 mM). Percentages underbars indicate the relative contribution of the two parameters to thetotal change in EPSP amplitude (see Methods).

120 mV

40 mV10 msec

FIG. 4. Facilitation by 5-HT under conditions of good voltage-clamp control in cultured cells. (A Left) Dependence of transmitterrelease on presynaptic step duration (from -50 mV to +12 mV) atshorter durations is not changed by 5-HT application: *, control; o,5-HT addition. (A Right) Similar experiment in another pair of cells.The EPSP elicited by the 4.5-msec step (from -40 mV to +34 mV)was above spike threshold after 5-HT addition. (B) Individual EPSPsfrom the same experiment as inA Right. Under voltage clamp, 5-HTproduced little facilitation: *, control; o, 5-HT addition. (C) Thesame cells as in B and A Right were recorded from 24 hr after 5-HTwashout, except that now EPSPs were elicited by presynaptic actionpotentials under current rather than voltage clamp. Facilitation wasconsiderably greater than it had been under voltage-clamp conditionsand was accompanied by spike broadening (C Right, lower records)and by an increase in the time-to-peak of the EPSP (C Right, upperrecords, A).

produced a 25% increase in action-potential duration, a 25%increase in the EPSP time-to-peak, and an 85% increase inEPSP amplitude. Thus, when the EPSP was not depressedand the presynaptic neuron was under optimal voltage-clampcontrol, the average facilitation induced by 5-HT was com-pletely blocked at short durations and substantially reducedeven at the longest duration, indicating that duration is thepredominant determinant of synaptic facilitation under theseconditions.

DISCUSSION

Action-Potential Broadening and Presynaptic Facilitation.We have found that transmitter release is a steep function ofduration of presynaptic depolarization and that minimizingthe contribution of spike broadening reduces facilitation.Moreover, facilitated synaptic potentials show increases intheir time-to-peak; these changes in the shape of the synapticpotential are similar to those produced when the actionpotential is broadened by K+-channel blockers or whendepolarizing commands under voltage clamp are prolonged.

A

BHigh Ca2+

5.)00CeC.

u4)

CeO

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8414 Neurobiology: Hochner et al.

These results, in conjunction with the finding that sensoryneuron action potentials broaden during facilitation, lead usto conclude that action-potential broadening contributesimportantly to the facilitation induced by both stimulation ofthe pathways that induce sensitization and application of themodulatory transmitter 5-HT. Recent evidence suggests thatclassical conditioning in the same reflex pathway involves thesame sensory neurons and an amplification of similar, if notidentical, ionic mechanisms (19).

Space Clamp Control and the Interpretation of Dura-tion-Release Curves. We imposed rectangular depolarizingsteps of increasing duration in the cell body and found thatsynaptic transmission was a steep but graded function of theduration of the presynaptic command. It is possible, none-theless, that the depolarization in the soma is distorted by thetime it reaches the terminals, so that the rectangular onset ofthe pulse in the soma appears as a charging curve at theterminals. Therefore, prolonging the pulse would give rise toa signal of greater amplitude at the releasing sites, andperhaps it is the increase in amplitude, rather than duration,that is responsible for the increase in transmitter release.Although we cannot entirely exclude this possibility in the

experiments on the intact ganglia, it is unlikely that thisexplanation can account for the input-output curves obtainedin the cultures. As an indication of the adequacy of ourvoltage clamp control in culture, we were able to show thatincreasing the amplitude of the step beyond about +30 mVcaused a decrease in the EPSP, as would be expected fromthe voltage dependence of the Ca2l current (6, 18), and alsothat the time-to-peak of the EPSP was well correlated withthe duration of the step.We have assumed that our rectangular voltage-clamp steps

are comparable to action potentials in eliciting transmitterrelease. This point has not been investigated in depth, but thefact that the amplitude and shape of EPSPs elicited withvoltage clamp are quite similar to those ofEPSPs elicited withaction potentials (Fig. 4 B and C) suggests that this assump-tion is not unreasonable.

Duration of the Voltage-Clamp Command and the Config-uration of the EPSP. That EPSPs elicited with action poten-tials show a prolongation of their rising phase during facili-tation is consistent with a broadening of the presynapticdepolarization in the terminals. However, there are otherpossible explanations for the increase in the time-to-peak ofthe EPSP that could account for these findings. For example,prolongation of transmitter release might result from aprolongation of the action of Ca2' inside the terminals. Thispossibility cannot be fully excluded, but the blockade offacilitation with short steps under voltage clamp arguesagainst it (Fig. 4).

Although our results indicate that broadening is an impor-tant contributor to facilitation when the synapse is notdepressed, they should not be taken to imply that broadeningis the only process contributing to facilitation under allcircumstances. Indeed, in the second paper of this series, wedescribe a second process that can affect transmitter releaseeven more profoundly than spike broadening. This processcomes powerfully into play when transmitter release isdepressed.

We thank Drs. J. Koester and C. F. Stevens for helpful sugges-tions; Drs. J. Koester, I. Kupfermann, S. Siegelbaum, and M. Spirafor comments on an earlier draft of the paper; H. Ayers and A.Krawetz for typing; and L. Katz, K. Hilten, and R. Woolley forpreparing the figures. S.S. was supported by a fellowship from theMcKnight Foundation.

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