release, and could account for much of the facilitation produced by sensitizing stimuli. when transmitter release is depressed, as occurs after the

Proc. Nati. cad. Sci. US Vol. 83, pp. 8410-8414, November 1986 Neurobiology ction-potential duration and the modulation of transmitter release from the sensory neurons of plysia in presynaptic facilitation and behavioral sensitization (K+-channel modulation/serotonin/learning/memory) INYMIN HOCHNER*, MRC KLEIN, SMUEL SCHCHER, ND ERIC R. KNDEL Howard Hughes Medical Institute, Center for Neurobiology and ehavior, Columbia University, College of Physicians and Surgeons, and The New York State Psychiatric Institute, New York, NY 10032 Contributed by Eric R. Kandel, July 16, 1986 STRCT Presynaptic facilitation of transmitter release from plysia sensory neurons is an important contributor to behavioral sensitization of the gill and siphon withdrawal reflex. The enhanced release is accompanied by reduction of the serotonin-sensitive S current in the sensory neurons and a consequent increase in duration of the presynaptic action potential (ranging from 10% to 30%). We find that changes of similar magnitude in the duration of depolarizing voltageclamp steps in sensory neurons in intact abdominal ganglia yield increases in synaptic potentials of 45-120%. In dissociated cell culture, these changes lead to increases of 25-60% in the synaptic potential. Prolongation of presynaptic depolarization using voltage clamp or prolongation of the duration of the action potential by K+-channel blockers leads to prolongation of the time-to-peak of the synaptic potentials; similar changes in time-to-peak occur during presynaptic facilitation. The time-to-peak is not changed by homosynaptic depression or by changing the Ca2' concentration, procedures that alter release without changing the duration of the action potential. Preventing the spike from broadening by voltage clamping the presynaptic neuron substantially reduces or blocks the facilitation. These results suggest that broadening of the action potential during facilitation is a causal factor in the enhancement of transmitter release. Short-term sensitization of the gill and siphon withdrawal reflex of plysia leads to facilitation of transmitter release from the presynaptic terminals of the sensory neurons onto their various target cells, the interneurons and the motor neurons (1). In earlier work, we described two changes that accompany presynaptic facilitation in sensory neurons: (i) a decrease in the S current, a serotonin (5-HT)-sensitive K+ current (2-5); and (ii) an increase in Ca2+ transients measured with the Ca2l indicator arsenazo III (6). We proposed that the decrease in K+ conductance augments transmitter release by prolonging 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). lthough the function of the alteration in Ca2` handling is not known, oyle et al. (6) suggested that it might act synergistically with the spike broadening to enhance transmitter release. This is the first of two related papers designed to analyze the relative contributions to presynaptic facilitation of spike broadening and other potential contributing processes, such as the altered handling of Ca2'. In this paper, we attempt to determine the degree to which spike broadening contributes to presynaptic facilitation. We find that broadening produced by facilitating stimuli has an important effect on transmitter The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. 1734 solely to indicate this fact. release, and could account for much of the facilitation produced by sensitizing stimuli. roadening is not, however, the only determinant of facilitation. In the subsequent paper (10), we show that an additional set of facilitatory processes becomes dominant when transmitter release is depressed, as occurs after the repeated activation of the sensory neurons that accompanies behavioral habituation. METHODS bdominal ganglia were removed from plysia californica weighing 100-250 g (Pacific iomarine, Venice, C; Sea Life Supply, Sand City, C). The ganglia were immersed for 45 sec in 0.5% glutaraldehyde [to prevent sheath contractions (11)] in artificial sea water [SW; 460 mm NaCl/10 mm KCl/11 mm CaCl2/55 mm MgCl2/10 mm Tris or Hepes (Sigma), ph 7.6], rinsed in SW, desheathed, and pinned out on Sylgard. In the experiments in culture, abdominal or pleural sensory neurons and followers were dissociated, plated, and grown by the method of Schacher and Proshansky (12). To prevent spiking in followers, they were held hyperpolarized. Voltage clamping was done with a Dagan 8500 two-microelectrode voltage clamp; microelectrodes were filled with 2.5 M KC1. Low-ionic-strength solution contained 50 mm NaCl, 10 mm KCl, 33 mm CaCl2, 25 mm tetraethylammonium chloride (Et4NCl, Kodak), 10 mm Tris, and 0.5 M sucrose (Sigma). For high-divalent-cation seawater, SW was modified to contain 260 mm NaCl, 60 mm CaCl2, and 140 mm MgCl2. Other compounds used were 5-HT-creatinine sulfate complex (Sigma) and 3,4-diaminopyridine (3,4-DP, Sigma). ll experiments were done at room temperature. The shapes of (excitatory) postsynaptic potentials [(E)PSPs] were analyzed with a Prowler laboratory computer (Norland, Ft. tkinson, WI). In some experiments EPSPs were averaged with a signal averager. To calculate the relative contributions to the changes in amplitude of changes in time-topeak (T) of the EPSP and in its rate of rise (R), we used the following formula: % contribution of T = log POS x 100 Tpre log TPOSt + log RPOSt Tpre Rpre where Xpre is the value of X before the procedure that altered EPSP amplitude and Xpost is the value afterwards. The bbreviations: 5-HT, serotonin; Et4N', tetraethylammonium; 3,4- DP, 3,4-diaminopyridine; (E)PSP, (excitatory) postsynaptic potential; SW, artificial sea water. *Present address: Department of Neurobiology, Life Sciences Institute, Hebrew University, Jerusalem, Israel. 8410

relative contribution of R was taken as 100 minus the percentage contribution of T. This formula was based on the simplifying assumption that the early part of the rising phase of the EPSP can be approximated by a straight line; the peak amplitude of the EPSP would then be equal to the product of R (the slope of the rising phase) and T. The ratios of the post values to the pre values were converted to logarithms so that the contributions of T and R could be expressed additively rather than as a product. The true amplitude is less than the product of R and T because the later part of the rising phase has a smaller slope than the earlier phase. s a result, the relative contributions of changes in T and R to alterations in amplitude should be considered as approximations only. RESULTS pplication of 5-HT, stimulation of the facilitating pathway from the head or tail, and injection of cmp or of the cmp-dependent protein kinase all produce an increase in the 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 intact abdominal ganglion sensory neurons (2), 15% in the cell bodies of abdominal ganglion sensory neurons in dissociated cell culture (15), and 25-30% in the cell bodies or growth cones of the pleural ganglion sensory neurons in vivo or in culture (14). Since these action potentials terminate substantially before the peak ofthe Ca2l current is reached, and since transmitter release is thought to vary exponentially with the Ca2l current (16), small changes in duration could have large effects on transmitter release. To determine if the amount of broadening actually observed contributes importantly to presynaptic facilitation, we performed experiments on abdominal sensory neurons, both in intact ganglia and in culture, and on pleural sensory neurons in culture. There Is a Steep Relationship etween the Duration of the Presynaptic Command Pulse and Transmitter Release in Undepressed Synapses. If prolongation of the duration of the action potential contributes significantly to presynaptic facilitation, and if the change in action potentials recorded in the cell body is indicative of what happens at the presynaptic terminals (see ref. 14), then dependence oftransmitter release on the duration of the action potential should be quite steep Neurobiology: Hochner et al Proc. NatL. cad. Sci. US 83 (1986) 8411 in the range of 2-3 msec, the normal duration of the sensory neuron spike (see also ref. 17). We first examined the dependence of release on the duration ofpresynaptic depolarization under voltage clamp in intact abdominal ganglia. We found that we obtained the best voltage-clamp control over release when we used solutions with a low ionic strength and added Et4N'. Under these conditions, transmitter release was graded as the depolarizing command was increased in amplitude. Small increases in pulse duration in the range of normal action potentials (about 2.0-3.0 msec) gave graded and relatively large increases in the amount of transmitter liberated from the terminals (Fig. 1). In the average of three experiments starting from initial durations of 2.5-3.0 msec, increases in duration of 10, 20, and 30%o produced increases in transmitter release of 45, 84, and 121%, respectively. Thus, the changes in duration produced by sensitizing stimuli in normal sea water could increase transmitter release substantially. To obtain better control of transmitter release in more normal bathing solutions, we carried out a series of experiments in which we examined the dependence of transmitter release on duration of presynaptic depolarization in dissociated cell cultures. y plating sensory neurons close to motor cells, we could shorten the distance from the cell body to the terminals and achieve better voltage control of the terminals without modifying the composition of the normal bathing solution. In dissociated cell cultures from pleural sensory cells, now in the presence of normal bathing solution, release again proved quite sensitive to changes in duration (Fig. i). In the average of three experiments starting from a step duration of 2 msec, increases in duration of 10, 20, and 30% enhanced transmitter release by 25, 46, and 62%, respectively. If the increases in duration that were seen in pleural cell bodies in culture (25-30%) were similar to those occurring in the terminals, as work on growth cones suggests (14), then the increases in action-potential duration produced by connective stimulation or 5-HT would contribute significantly to presynaptic facilitation. lterations in Duration of the ction Potential Produce Predictable Changes in the Shape of the EPSP. Our conclusions about what happens in the presynaptic terminals rest in large part on indirect arguments based on observations in the cell body. To gain more direct access to events at the 3.0 3.5 7 r 4 - E 2 4.0 4.5 5.0 5.5 mse C Post J 2.5 mv 0.5 sec X 1 02.5 5 7.5 10 -F v I,.-.I Pre +12mVI Vm -50 mv J V- m 5 msec 10 mv 1100 n l 20 E 15 8o 10 1 2 3 4 5 6 7 8 9 10 FIG. 1. Dependence of transmitter release on presynaptic step duration. () sensory neuron in an intact abdominal ganglion was voltage-clamped at -50 mv in low-ionic-strength bathing solution, and the voltage was stepped to +40 mv. PSPs of different amplitudes were elicited as the step was changed in duration. The top part of the figure shows samples of the data from which this plot was constructed. ( 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 progressively larger EPSPs in a follower neuron (Post). ( Right) Plot of the dependence of transmitter release on presynaptic step duration (same cell as in Left). Im. membrane current; Vm, membrane voltage. athing medium was normal SW. The curves in this and all subsequent figures were drawn by eye. *

8412 Neurobiology: Hochner et al. terminals, we tried to infer some of the properties of the presynaptic depolarization from observation of the shape of the postsynaptic response. We noted, as have Llinas et al. (18), that when a neuron is voltage-clamped and the depolarizing command is prolonged well past the release threshold, the time-to-peak of the postsynaptic potential is progressively increased (Fig. 1 Left and 2). Similar changes in time-to-peak occurred when the spike was broadened as a result of application of the K+-channel blockers Et4N' or 3,4-DP. Fig. 2 Right illustrates the broadening of the action potential and the increase and prolongation of transmitter release caused by 3,4-DP. y contrast, when synaptic transmission was altered without changing the duration of the spike, as by bathing in medium containing different Ca2l concentrations or during homosynaptic depression, the shape of the PSP was unchanged (Figs. 2 Left, 3 Top and Middle, and 3). The absence of a shape change With altered Ca2' and with homosynaptic depression implies that the absolute level of transmitter release can be altered substantially without any necessary concomitant change in the shape of the PSP. Since the somatic action potential broadens during facilitation, finding that the facilitated EPSP is also prolonged would support the idea that prolongation of the action potential also occurs in the presynaptic terminals and that this broadening is causally related to presynaptic facilitation.. Presynaptic Facilitation Is ccompanied by Changes in the Shape of the PSP. We therefore examined synaptic potentials in intact ganglia before and after stimulation of a facilitatory pathway. We found a marked prolongation of the rising phase of the EPSP (Fig. 3 Top and Middle). The average increase in time-to-peak in normal seawater was 18% ± 7 (n = 6) and 43% ± 18 (n = 6) in high-divalent-cation medium. lthough the time-to-peak changed significantly, the later decay phase was not changed, making it unlikely that a change in passive properties of the postsynaptic membrane is responsible for the altered configuration of the synaptic potentials (Fig. 3 ottom). Direct measurement of membrane resistance in other experiments also revealed no change. Thus, the prolongation of the EPSP that accompanies presynaptic facilitation is consistent with a prolongation in transmitter release from the presynaptic site. s pointed out above, there is little or no change in the EPSP configuration during homosynaptic depression (Fig. 3 Top and Middle and 3). This suggests that depression and facilitation diverge in mechanism at some point. Prevention of Spike roadening Reduces Presynaptic Facilitation. The experiments we have considered so far are consistent with a mechanism for presynaptic facilitation that Post Pre +12 mv -l -m I -50 mv?, I 5 msec 10 mv involves broadening of the action potential as one important component. However, our data do not allow us to exclude contributions of other components to this facilitation. This question is particularly relevant because earlier we had found a second, independent effect associated with presynaptic facilitation: the Ca2l concentration transient caused by a depolarizing step is directly modulated by 5-HT (6). This suggests that the cell's handling of Ca2l is altered and might be involved in facilitation of transmitter release. Since Ca2+ handling may involve intracellular processes, it is possible that changes in such a process might not be detected by examining the membrane currents alone. Nevertheless, if the change in K+ conductance is important in facilitation, a causal connection between the K+ conductance decreases and facilitation should be demonstrable. The only obvious way that a K+ conductance decrease could be translated into increased transmitter release is by means of an effect on the membrane potential or the action potential. This effect might be a change in the steady-state potential, enhancement of a passively conducted depolarization, or a change in the number or configuration of action potentials (9). Preventing potential changes from occurring by means of an adequate voltage clamp of the sensory neuron terminals should then reduce facilitation, despite the fact that the 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 a manher that would be interpretable. However, in culture, we were in some cases able to elicit graded transmitter release from a presynaptic neuron under conditions of good voltage control and to examine the effects of 5-HT. Fig. 4 Left illustrates the characteristic duration-release curve under voltage clamp. Later in the experiment, 5-HT was applied, and a curve was again generated. The curve obtained after the application of 5-HT was superimposable on the control curve, except for the longest pulse duration (). This superimposability suggests that when the synaptic potential is not depressed, the input-output curve is not significantly affected by 5-HT except at the longest duration. Under these circumstances, processes other than spike broadening appear to contribute little to facilitation. Fig. 4 Right and illustrate a more limited input-output curve in an experiment in which voltage-clamp and currentclamp experiments could be compared in the same cell. n input-output curve was first generated under voltage clamp. s before, adding 5-HT did not significantly affect the amplitude of the EPSP at any duration except perhaps the longest. The experiment was then repeated on the same cell the next day under current clamp (Fig. 4C). 5-HT now 400t Proc. Natl. cad. Sci. US 83 (1986) FIG. 2. Effect of duration of the presynaptic depolarization on the shape of the EPSP. The two parameters that determine the amplitude of an EPSP are its rate-of-rise (or initial slope of its rising phase) and its time-to-peak amplitude (see Fig. 3 Inset). Increasing either of these will increase EPSP amplitude. () Once the threshold for the EPSP has been exceeded, increasing the duration of presynaptic voltage-clamp depolarizations leads to an increase in the time-to-peak of the EPSP () without affecting the initial rate-of-rise, or slope. ( Left) During homosynaptic depression, there is no change in the presynaptic action potential (bottom trace) and the time-to-peak of the EPSP remains constant, whereas the rate-of-rise decreases (top traces). ( Right) roadening the presynaptic action potential with the K+-channel blocker 3,4-DP (0.1 mm) (bottom traces) leads to an increase in EPSP time-to-peak (top traces, ) without changing the rate-of-rise. 2 mv I50 mv

5.) 00 Ce C. u4) CeO Neurobiology: Hochner et al High Ca2+ I! PSP amplitude Rate-of-rise Time-to-peak Homosynaptic depression Heterosynapti:C facilitation 110 mv 41m2 mv 1.6 mv 40 msec 3,4-DP FIG. 3. Changes in the shape of the EPSP accompanying presynaptic facilitation. ( Top) Superimposition of the first EPSP in a homosynaptic depression series (largest EPSP) and two EPSPs later on. There is relatively little change in time-to-peak during homosynaptic depression (). ( Middle) The smallest EPSP is one ofthe depressed EPSPs from Top, while the two larger EPSPs were elicited after presynaptic facilitation induced by connective stimulation. In the facilitated EPSPs, the time-to-peak is substantially increased. athing medium contained high amounts of divalent cations. ( ottom) In another sensory neuron, EPSPs from cells bathing in normal SW were elicited before and after connective stimulation, and the decay phases were compared. Twenty EPSPs before and 20 after connective stimulation were averaged, and the two averages were normalized with respect to amplitude. The average of the facilitated group was somewhat larger and longer than that of the control group, yet shifting this record to the left shows that the decay phases are essentially superimposable. This suggests that the change in EPSP duration cannot be accounted for by a change in passive postsynaptic membrane properties. () These histograms compare the relative contributions of rate-of-rise and time-to-peak changes in EPSP amplitude under several conditions: (i) change in extracellular Cae' concentration; (ii) homosynaptic depression; (iil) heterosynaptic facilitation caused by connective stimulation; and (iv) blockade of K+ channels with 3,4-DP (0.1 mm). Percentages under bars indicate the relative contribution of the two parameters to the total change in EPSP amplitude (see Methods). 0% ~.. ;2 *1 =i, cd Cu4 Proc. Natl. cad. Sci. US 83 (1986) 8413 0 50 0 > E 40 [ 0 30 D to o0~~~~~l ~~~~~~~~~E 20 CIO IX 10 0 2 4 6 8 10 Voltage clamp C 1 msec Post Pre 40 0X Spike t 1 2 3 4 5 1.5 msec 2.1 msec 2.6 msec 5-HT 4 Current clamp 20 mv 120 mv 40 mv FIG. 4. Facilitation by 5-HT under conditions of good voltageclamp control in cultured cells. ( Left) Dependence of transmitter release on presynaptic step duration (from -50 mv to +12 mv) at shorter durations is not changed by 5-HT application: *, control; o, 5-HT addition. ( 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. () Individual EPSPs from the same experiment as in Right. Under voltage clamp, 5-HT produced little facilitation: *, control; o, 5-HT addition. (C) The same cells as in and Right were recorded from 24 hr after 5-HT washout, except that now EPSPs were elicited by presynaptic action potentials under current rather than voltage clamp. Facilitation was considerably greater than it had been under voltage-clamp conditions and was accompanied by spike broadening (C Right, lower records) and by an increase in the time-to-peak of the EPSP (C Right, upper records, ). produced a 25% increase in action-potential duration, a 25% increase in the EPSP time-to-peak, and an 85% increase in EPSP amplitude. Thus, when the EPSP was not depressed and the presynaptic neuron was under optimal voltage-clamp control, the average facilitation induced by 5-HT was completely blocked at short durations and substantially reduced even at the longest duration, indicating that duration is the predominant determinant of synaptic facilitation under these conditions. DISCUSSION ction-potential roadening and Presynaptic Facilitation. We have found that transmitter release is a steep function of duration of presynaptic depolarization and that minimizing the contribution of spike broadening reduces facilitation. Moreover, facilitated synaptic potentials show increases in their time-to-peak; these changes in the shape of the synaptic potential are similar to those produced when the action potential is broadened by K+-channel blockers or when depolarizing commands under voltage clamp are prolonged.

8414 Neurobiology: Hochner et al. These results, in conjunction with the finding that sensory neuron action potentials broaden during facilitation, lead us to conclude that action-potential broadening contributes importantly to the facilitation induced by both stimulation of the pathways that induce sensitization and application of the modulatory transmitter 5-HT. Recent evidence suggests that classical conditioning in the same reflex pathway involves the same sensory neurons and an amplification of similar, if not identical, ionic mechanisms (19). Space Clamp Control and the Interpretation of Duration-Release Curves. We imposed rectangular depolarizing steps of increasing duration in the cell body and found that synaptic transmission was a steep but graded function of the duration of the presynaptic command. It is possible, nonetheless, that the depolarization in the soma is distorted by the time it reaches the terminals, so that the rectangular onset of the pulse in the soma appears as a charging curve at the terminals. Therefore, prolonging the pulse would give rise to a signal of greater amplitude at the releasing sites, and perhaps it is the increase in amplitude, rather than duration, that is responsible for the increase in transmitter release. lthough we cannot entirely exclude this possibility in the experiments on the intact ganglia, it is unlikely that this explanation can account for the input-output curves obtained in the cultures. s an indication of the adequacy of our voltage clamp control in culture, we were able to show that increasing the amplitude of the step beyond about +30 mv caused a decrease in the EPSP, as would be expected from the voltage dependence of the Ca2l current (6, 18), and also that the time-to-peak of the EPSP was well correlated with the duration of the step. We have assumed that our rectangular voltage-clamp steps are comparable to action potentials in eliciting transmitter release. This point has not been investigated in depth, but the fact that the amplitude and shape of EPSPs elicited with voltage clamp are quite similar to those of EPSPs elicited with action potentials (Fig. 4 and C) suggests that this assumption is not unreasonable. Duration of the Voltage-Clamp Command and the Configuration of the EPSP. That EPSPs elicited with action potentials show a prolongation of their rising phase during facilitation is consistent with a broadening of the presynaptic depolarization in the terminals. However, there are other possible explanations for the increase in the time-to-peak of the EPSP that could account for these findings. For example, prolongation of transmitter release might result from a prolongation of the action of Ca2' inside the terminals. This possibility cannot be fully excluded, but the blockade of facilitation with short steps under voltage clamp argues against it (Fig. 4). Proc. Natl. cad. Sci. US 83 (1980) lthough our results indicate that broadening is an important contributor to facilitation when the synapse is not depressed, they should not be taken to imply that broadening is the only process contributing to facilitation under all circumstances. Indeed, in the second paper of this series, we describe a second process that can affect transmitter release even more profoundly than spike broadening. This process comes powerfully into play when transmitter release is depressed. We thank Drs. J. Koester and C. F. Stevens for helpful suggestions; Drs. J. Koester, I. Kupfermann, S. Siegelbaum, and M. Spira for comments on an earlier draft of the paper; H. yers and. Krawetz for typing; and L. Katz, K. Hilten, and R. Woolley for preparing the figures. S.S. was supported by a fellowship from the McKnight Foundation. 1. Castellucci, V. & Kandel, E. R. (1976) Science 194, 1176-1178. 2. Klein, M. & Kandel, E. R. (1980) Proc. Natl. cad. Sci. US 77, 7492-7496. 3. Klein, M., Camardo, J. 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R. & Siegelbaum, S.. (1986) in Molecular spects of Neurobiology, eds. Levi-Montalcini, R., Calissano, P., Kandel, E. R. & Maggi,. (Springer, erlin), pp. 15-18. 15. Rayport, S. G. & Schacher, S. (1986) J. Neurosci. 6, 759-763. 16. ugustine, G. J., Charlton, M. P. & Smith, S. J. (1985) J. Physiol. (London) 369, 163-181. 17. Gingrich, K. J. & yrne, J. H. (1985) J. Neurophysiol. 53, 652-669. 18. Llinas, R., Steinberg, I. Z. & Walton, K. (1981) iophys. J. 33, 323-352. 19. Hawkins, R. D., brams, T. W., Carew, T. J. & Kandel, E. R. (1983) Science 219, 400-415.