Transforming Tonic Firing Into a Rhythmic Output in the ... › 3d84 › 9809451f5cf9f762d86754b… · Transforming Tonic Firing Into a Rhythmic Output in the Aplysia Feeding System:

Transforming Tonic Firing Into a Rhythmic Output in the Aplysia FeedingSystem: Presynaptic Inhibition of a Command-Like Neuronby a CPG Element

Itay Hurwitz,1,2,3 Abraham J. Susswein,1,2 and Klaudiusz R. Weiss3

1The Leslie and Susan Gonda (Goldschmied) Multidisciplinary Brain Research Center and 2Faculty of Life Sciences, Bar Ilan University,Ramat Gan, Israel; and 3Department of Physiology and Biophysics, Mount Sinai School of Medicine,New York City, New York

Submitted 28 May 2004; accepted in final form 1 August 2004

Hurwitz, Itay, Abraham J. Susswein, and Klaudiusz R. Weiss.Transforming tonic firing into a rhythmic output in the Aplysiafeeding system: presynaptic inhibition of a command-like neuron bya CPG element. J Neurophysiol 93: 829–842, 2005. First publishedAugust 11, 2004; doi:10.1152/jn.00559.2004. Tonic stimuli can elicitrhythmic responses. The neural circuit underlying Aplysia californicaconsummatory feeding was used to examine how a maintained stim-ulus elicits repetitive, rhythmic movements. The command-like cere-bral-buccal interneuron 2 (CBI-2) is excited by tonic food stimuli butinitiates rhythmic consummatory responses by exciting only protrac-tion-phase neurons, which then excite retraction-phase neurons after adelay. CBI-2 is inhibited during retraction, generally preventing itfrom exciting protraction-phase neurons during retraction. We havefound that depolarizing CBI-2 during retraction overcomes the inhi-bition and causes CBI-2 to fire, potentially leading CBI-2 to exciteprotraction-phase neurons during retraction. However, CBI-2 synapticoutputs to protraction-phase neurons were blocked during retraction,thereby preventing excitation during retraction. The block was causedby presynaptic inhibition of CBI-2 by a key buccal ganglion retrac-tion-phase interneuron, B64, which also causes postsynaptic inhibi-tion of protraction-phase neurons. Pre- and postsynaptic inhibitioncould be separated. First, only presynaptic inhibition affected facili-tation of excitatory postsynaptic potentials (EPSPs) from CBI-2 to itsfollowers. Second, a newly identified neuron, B54, produced postsyn-aptic inhibition similar to that of B64 but did not cause presynapticinhibition. Third, in some target neurons B64 produced only presyn-aptic but not postsynaptic inhibition. Blocking CBI-2 transmitterrelease in the buccal ganglia during retraction functions to preventCBI-2 from driving protraction-phase neurons during retraction andregulates the facilitation of the CBI-2 induced EPSPs in protraction-phase neurons.

I N T R O D U C T I O N

A tonic stimulus can elicit rhythmic movements (Marder andCalabrese 1996; Pearson and Gordon 2000). Neural circuitsgiving rise to such movements must contain mechanisms fortransforming a tonic stimulus into a rhythmic response. Thepresent report uses the feeding behavior in the marine gastro-pod mollusk Aplysia to examine some of the mechanisms thattransform a tonic stimulus into rhythmic, repetitive responses.

Aplysia consummatory feeding behaviors are elicited byfood touched to the lips or stimulating the interior of the mouth

(Kupfermann 1974). Tonically maintained stimuli elicit re-peated, rhythmic protraction and retraction movements of thetoothed radula (Kupfermann 1974; Morton and Chiel 1993a,b).Buccal motor programs corresponding to protraction and re-traction movements in the intact animal can be monitored frommany neurons within the buccal ganglia as well as via extra-cellular recordings from buccal ganglia nerves (Church andLloyd 1994; Hurwitz and Susswein 1996; Morton and Chiel1993a,b). Buccal motor programs are organized by a centralpattern generator (CPG) containing separate interneurons thatdrive the protraction and retraction phases (Hurwitz and Sus-swein 1996; Hurwitz et al. 1997; Plummer and Kirk 1990).CPG activity is initiated via command-like neurons. The mostprominent such neuron, cerebral-buccal interneuron 2 (CBI-2),is excited by food stimulating the lips and monosynapticallyexcites protraction-phase interneurons, thereby initiating theiractivity (Hurwitz et al. 2003). Similar to the effects of tonicfood stimulation on behavior, tonic depolarization of CBI-2causes repeated, rhythmic buccal motor programs that ceasewhen CBI-2 stimulation is terminated.

How are tonic stimuli to the lips or to CBI-2 converted intorhythmic buccal motor programs? Previous studies have iden-tified two such mechanisms. One arises from reciprocal inhi-bition within the CPG. Activity in protraction-phase neuronsinitiates activity, after a delay, in retraction-phase neurons,which in turn inhibit protraction-phase neurons and therebyturn off protraction while retraction progresses. A secondmechanism arises from a feedback loop from the CPG to thecommand-like CBI-2 neuron. CBI-2 is inhibited during theretraction phase of a buccal motor program, thereby reducingits likelihood to fire during the retraction phase (Hurwitz et al.1999b; Rosen et al. 1991). Because of the recurrent inhibitionfrom retraction-phase neurons, activity of CBI-2 in response toa tonic stimulus is itself phasic, and CBI-2 generally fires andexcites protraction-phase interneurons only during the protrac-tion phase. However, these two mechanisms are unlikely to besufficient to ensure that tonic stimuli elicit a rhythmic buccalmotor program. Strong inputs to CBI-2 that depolarize itsufficiently during the retraction phase will override the inhi-bition and elicit firing. CBI-2 firing during retraction wouldthen excite and perhaps cause firing in protraction-phase neu-rons during the retraction phase.

Address for reprint requests and other correspondence: I. Hurwitz, Interdis-ciplinary Program in the Brain Sciences, Bar-Ilan University, Ramat Gan52900, Israel (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

J Neurophysiol 93: 829–842, 2005.First published August 11, 2004; doi:10.1152/jn.00559.2004.

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This study examined the consequences of driving CBI-2 tofire during the retraction phase. We found that firing CBI-2during retraction had no effects on buccal motor programsbecause a third mechanism is present that contributes to theconversion of a tonic stimulus to CBI-2 into a phasic response.In addition to inhibiting all of the protraction-phase neurons,the major retraction-phase interneuron B64 also causes pow-erful presynaptic inhibition of CBI-2, thereby blocking itseffects in the buccal ganglia and preventing it from drivingprotraction-phase neurons during the retraction phase.

M E T H O D S

The experimental subjects were Aplysia californica weighing 150–300 g provided by Marinus (Long Beach, CA) and by the NationalResource for Aplysia at the University of Miami. Animals weremaintained at 14–16°C in holding tanks containing aerated, filteredseawater. Before being dissected, animals were anesthetized andimmobilized by injection with isotonic MgCl2 (50% of body wt). Thebuccal and cerebral ganglia were removed with the cerebral-buccalconnectives (CBCs) intact. The cerebral and buccal ganglia werepinned to the floor of a recording chamber with the ventral surface ofthe cerebral ganglion and the caudal surface of the buccal gangliafacing up. The sheath overlying the surface of the ganglia wasremoved using ultrafine scissors.

Cerebral and buccal ganglia circuitry

Tonic depolarization of the command-like CBI-2 neurons inducesrepeated buccal motor programs (BMPs) that are organized by thebuccal ganglion CPG (Church and Lloyd 1994; Hurwitz et al.1999a,b; Perrins and Weiss 1998; Rosen et al. 1991). The ability ofCBI-2 to elicit BMPs arises in part from the large, facilitating fastexcitatory postsynaptic potentials (EPSPs) that CBI-2 firing induces inthe protraction-phase interneurons that are part of the CPG (Hurwitzet al. 2003; Sanchez and Kirk 2000). CBI-2 also induces slow EPSPsin the protraction-phase interneurons (Hurwitz et al. 1999a). Buccalganglia protraction-phase neurons are excited by CBI-2. The presentstudy examined a number of protraction-phase neurons that areexcited by CBI-2, such as B31/B32, B34, B61/B62, and B63 (Fig.1A). Previous studies have indicated that activity in most of theprotraction-phase neurons is not necessary to elicit a buccal motorprogram because preventing them from firing does not abolish theability of the ganglion to express a program (Hurwitz et al. 2003).Exceptions are the electrically coupled B31/B32 and B63 neurons,which are always active during buccal motor programs. In addition,hyperpolarizing them can block the expression of a program. Theability of CBI-2 to initiate buccal motor programs is largely explainedby its strong, facilitating excitation of B63 (Hurwitz et al. 2003).

The retraction phase of a buccal motor program is triggered byfiring in neuron B64 (Hurwitz and Susswein 1996). Additional inter-neurons have important functions in retraction (Plummer and Kirk1990), but they are not always active during the expression of a buccalmotor program (Nargeot et al. 2002), whereas B64 firing is aninvariant feature of a buccal motor program. B64 activity is initiatedas a result of activity in the protraction-phase neurons via their effectson an unidentified element of the CPG that has been called the “z” cell(Baxter et al. 1997; Hurwitz and Susswein 1996). The existence of thez cell has been inferred by modeling studies (Baxter et al. 1997) aswell as via its effects on B64. B64 displays a prominent, endogenousplateau potential: a brief depolarization induces a burst of spikes thatlong outlasts the stimulus. Firing in B64 produces a large inhibitorypostsynaptic potential (IPSP) in protraction-phase neurons and depo-larizes and caused firing in other retraction-phase neurons (Hurwitzand Susswein 1996).

Recording apparatus and bathing solutions

Preparations were bathed in artificial seawater [ASW, which con-tained (in mM): 460 NaCl, 10 KCl, 11 CaCl2, 55 MgCl2, and 5NaHCO3] at pH � 7.64. In some experiments, the buccal and cerebralganglia were placed in a solution containing an increased concentra-tion of divalent cations [HiDi saline containing (in mM) 311 NaCl, 9KCl, 33 CaCl2, 132 MgCl2, and 5 NaHCO3] to reduce polysynapticactivity of coupled neurons and follower neurons (Hurwitz et al.2000). Except for experiments using voltage clamping, intracellularrecordings were obtained from isolated ganglia preparations main-tained at room temperature (18–22°C). Most experiments were per-formed under continuous fluid exchange, using a peristaltic pump at arate of 10% volume/min.

Intracellular recordings were made from CBI-2 as well as frombuccal interneurons B64, B54, B63, and B34, and the buccal protrac-tion-muscle (I2) motoneurons B31/B32 and B61/B62. Other neuronsthat were often recorded include radula closure motor neuron B8 andB4/B5. The neurons were identified by previously established mor-phological and/or physiological criteria (Gardner and Kandel 1977;Hurwitz and Susswein 1996; Hurwitz et al. 1994, 1996, 1999b;Jahan-Parwar et al. 1983; Morton and Chiel 1993a,b; Rosen et al.1991; Susswein and Byrne 1988).

To generate an action potential in a neuron, 20-ms depolarizingcurrent pulses were injected, and the appearance of one-for-one actionpotentials was monitored. CBI-2 was tonically fired at 7–20 Hz for aperiod that did not exceed 3 min. Repeated stimulus sets to CBI-2were separated by 10-min intervals.

Intracellular recording, and stimulation

For intracellular recording and stimulation, neurons were impaledwith single-barreled microelectrodes that were made of thin-walledglass tubing that was filled with 1.9 M potassium acetate and 0.1 Mpotassium chloride. The electrodes were pulled so that their imped-ances ranged from 10 to 15 M�, and following beveling, they hadfinal resistances of 6–10 M�. The activity of up to four neurons wasmonitored via intracellular recording using conventional electrome-ters. The extracellular activity in up to two nerves was also monitored.A Grass stimulator (S88) controlled the intracellular stimuli deliveredto the neurons.

Voltage clamping

Voltage clamping was performed at 17°C using 1–5 M� electrodesfilled with 1 M KCl. Experiments were performed in an 0.5-mlchamber. The currents and voltages were recorded with an Axoclamp2 (Axon Instruments) current/voltage clamp that was controlled by acomputer running the Clampex component of pClamp 8.0 (AxonInstruments). Data were digitized and recorded using this program,via a Digidata 1200A digitizer (Axon Instruments). Preliminary dataanalyses used the Clampfit component of pClamp 8.0.

R E S U L T S

In Aplysia, a CPG in the buccal ganglia drives repeated cyclesof radula protraction and retraction (Figs. 1, B and C, and 2A). Thebuccal CPG is composed of mutually inhibitory protraction andretraction-phase interneurons (Figs. 1, B and C, and 2A). There islittle or no overlap in the firing of protraction phase (e.g., B31/B32, B34, B61/B62, and B63), and retraction-phase (e.g., B64)interneurons and motor neurons (Hurwitz et al. 1997). A smallpopulation of additional neurons (e.g., B4/B5, the B8s) may shiftthe degree to which they are active in the two phases (Fig. 1D, 1and 2). In the isolated buccal ganglia, activation of the CPGcauses organized BMPs, which correspond to protraction and

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retraction movements in the intact animal (Hurwitz et al. 1996,1997). CBI-2 elicits monosynaptic facilitating EPSPs in protrac-tion-phase neurons, thereby initiating the protraction phase of aBMP (Hurwitz et al. 2003). After a delay of several seconds,retraction-phase interneurons are activated (Hurwitz et al. 1997).The firing pattern of CBI-2 is similar to that of the protraction-phase interneurons: it fires in phase with buccal ganglia protrac-tion-phase neurons (although CBI-2 activity slightly precedesbuccal ganglia activity), and the firing is suppressed during re-traction phase of a BMP in part via buccal to cerebral interneurons(Hurwitz et al. 1999b; Rosen et al. 1991). The inhibition of CBI-2during retraction would be functionally adaptive because firing ofCBI-2 during retraction might cause protraction-phase interneu-rons and motor neurons to fire during the retraction phase. Thesimultaneous firing of the protraction and retraction muscleswould interfere with the retraction movement.

Similar motor programs are generated by phasic and tonicfiring of CBI-2

Although CBI-2 is inhibited during the retraction phase, theinhibition can be overcome by depolarizing stimuli. We examinedthe possible consequences on BMPs of firing CBI-2 during theretraction phase (Fig. 2). Firing in CBI-2 was regulated so that itfired at a constant rate during both the protraction and retractionphases. In this experiment (n � 5), the protraction phase wasmonitored by intracellular recordings from protraction-phase in-terneuron B63 as well as by extracellular recordings from the I2nerve, which contains axons of the protraction-phase neuronsB31/B32 and B61/B62 that drive contractions of the major pro-traction muscle, I2 (Hurwitz et al. 1994, 1996, 2000). The retrac-tion phase was monitored via intracellular recordings from neuronB4, which fires during retraction, as well as via intracellular

FIG. 1. Cerebral-buccal interneuron 2 (CBI-2) drivesa central pattern generator (CPG) organizing repetitiveprotraction-retraction movements located in the buccalganglia. A: a map of the rostral (right) and caudal (left)surfaces of the buccal ganglia indicating the positions ofthe neurons discussed in this paper. B: a schematicdiagram showing the relationships between command-like neuron CBI-2 and the protraction and retraction-phase neurons in the buccal ganglia. CBI-2 drives thebuccal ganglia CPG by exciting protraction-phase neu-rons. The protraction-phase neurons in turn drive retrac-tion-phase neurons after a delay via a polysynapticpathway the elements of which are incompletely char-acterized. Protraction and retraction-phase neurons aremutually inhibitory. Retraction-phase neurons also in-hibit CBI-2. In this and in C, triangles represent excita-tion and circles represent inhibition. The size of thetriangles and circles are roughly proportional to theamplitude of the synaptic connections. A resistor repre-sents an electrical synapse, and a dashed line representsa polysynaptic connection. C: a diagram showing thesynaptic relationships among the neurons discussed inthis paper. CBI-2 monosynaptically excites protraction-phase CPG elements B63, B34, and B31/B32. Theprotraction-phase neurons are mutually excitatory. B64is a major interneuron driving retraction. It stronglyinhibits all of the protraction-phase interneurons and isitself weakly inhibited by them. D: firing patterns duringa buccal motor program of the various neurons dis-cussed in the paper. A darker area represents an in-creased firing. Asterisks, CPG elements.

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recordings from motor neuron B8 and extracellular recordingsfrom the radula nerve. B8 and radula nerve activity are seenduring both protraction and retraction. The activity that is main-tained after the end of protraction was utilized for monitoringretraction phases. BMPs were initiated via continuous stimulationof CBI-2 at 17 Hz with brief current pulses that evoked only asingle spike in CBI-2.

When CBI-2 was injected with 18-nA current pulses (Fig.2A1), spikes were initiated in CBI-2. The spikes elicited summat-ing and facilitating monosynaptic EPSPs in B63. When theamplitude of the EPSPs exceeded threshold, B63 began to fire andBMPs were elicited as has been described previously (Hurwitz etal. 2003). The 18-nA current pulses that were sufficient to initiatespikes in CBI-2 prior to and during the protraction phase did notinitiate spikes during the retraction phase (Fig. 2A2) presumablybecause spikes were blocked by the phasic inhibitory input thatCBI-2 received during retraction.

Raising the amplitude of the current pulses in CBI-2 from 18to 25 nA (Fig. 2B1) overcame the inhibition that CBI-2received during retraction and elicited spikes during bothphases of the BMP (Fig. 2B2). However, the patterns ofactivity in the buccal ganglia were remarkably similar to thoseelicited when CBI-2 was fired only during protraction (com-pare Fig. 2, A1 and B1), in spite of the additional firing of

CBI-2 during retraction. In addition, no EPSPs were observedin B63 when CBI-2 was fired during the retraction phase.

Previous experiments have shown that CBI-2 elicits facili-tating EPSPs in a number of protraction-phase neurons (seeFig. 1C), including B34, B63, B31, and B61 (Hurwitz et al.2003; Sanchez and Kirk 2000). We examined the ability ofCBI-2 to elicit EPSPs in a variety of protraction-phase neuronsat different phases of a BMP (n � 26). Figure 3 illustrates thata train of spikes in CBI-2 elicited facilitating EPSPs in B63,B34, and B31 (see fast sweep in Fig. 3B). These eventuallysummated, causing a BMP (Fig. 3A). However, a train at thesame frequency (14 Hz) delivered during the retraction phasecompletely failed to elicit detectable EPSPs (see the fast sweepin Fig. 3C). Later trains, which occurred during the late portionof the retraction phase or following the end of the retractionphase, elicited EPSPs of progressively increasing amplitudewithin the bursts and between them (Fig. 3C).

These data indicate that when CBI-2 is fired early in theretraction phase, some mechanism completely suppressesCBI-2 elicited EPSPs in protraction-phase neurons. The sup-pression gradually declines through the later portion of theretraction phase and subsequent to retraction. The suppressionof CBI-2 elicited EPSPs is likely to explain why CBI-2 firingduring retraction has little or no effect on BMPs. This suppres-

FIG. 2. Tonic and phasic firing of CBI-2 generate similarcycles of biphasic motor program. The protraction phasewas monitored by an intracellular recording from neuronB63 and by an extracellular recording from the I2 nerve(I2N). The retraction phase was monitored by intracellularrecordings from neurons B4 and B8 and by an extracellularrecording from the radula nerve (RN). The activity thatoutlasts the protraction phase denotes retraction. A1: intra-cellular stimulation of CBI-2 with brief 18-nA currentpulses drives a repetitive motor program. During the retrac-tion phase of the motor program, CBI-2 received inhibitoryinput that blocked the initiation of action potentials. B1:intracellular stimulation of CBI-2 with larger (25 nA) cur-rent pulses drives a similar buccal motor program (BMP),although the neuron was spiking continuously. The lack offiring during the retraction phase (A2), in contrast to thecontinuous firing (B2), is clearly seen in the fast sweeprecordings of CBI-2 during the protraction and retractionphases of the motor program. The facilitated excitatorypostsynaptic potentials (EPSPs) develop to an amplitude of�10 mV (see arrow in A1), and no EPSPs were detectedduring the retraction phase (see arrow in B1).



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sion could be caused by either presynaptic or postsynapticinhibition.

Postsynaptic inhibition cannot suppress CBI-2elicited EPSPs

Protraction-phase neurons, including B34, B31/B32, B63,and B61/B62, are strongly inhibited during the retraction phaseby a large IPSP mediated via a conductance increase (Hurwitzet al. 1994, 1997). In principle, this IPSP could account for thesuppression of EPSPs from CBI-2 to protraction-phase neuronsduring retraction. If the conductance increase was sufficientlylarge, it could shunt completely the CBI-2-elicited EPSP. Wetested this possibility by examining the conductance increaseselicited in the protraction-phase neurons during the retractionphase. In this experiment, EPSPs in protraction neurons weresimulated by intracellular depolarizing current pulses.

Firing CBI-2 was used to elicit BMPs (n � 11). CBI-2stimulation was terminated just after the onset of the retractionphase, and sometime later two identical currents pulses wereinjected into a variety of protraction-phase neurons. One pulsewas delivered during the retraction phase, and the other wasdelivered a number of seconds after the retraction phase hadended. The voltage change during the retraction phase wasdecreased by somewhat �50% with respect to the voltagechange after the retraction phase (Fig. 4). These data indicatethat the conductance change caused by the IPSP that underliesretraction would shunt a CBI-2-elicited EPSP in protraction-phase neurons, thereby lowering its amplitude, but the shuntingwould be insufficient to block the EPSP completely.

To examine quantitatively whether the retraction causes aconductance change that is sufficient to shunt CBI-2-inducedEPSPs, protraction-phase neuron B31/B32 was voltageclamped (Fig. 5). The neuron was held at �60 mV, its restingpotential. Either 4- or 8-s sweeps were observed at a frequencyof once per 30 s. Voltage steps were applied in �10-mVincrements, until 0 mV, and the steps were maintained for mostof the sweep. Many records showed spontaneous intermittentslow inward deflections followed by outward deflections. Incurrent-clamp conditions, the inward currents would underliedepolarizations of B31/B32 during protraction, and the out-

ward currents would underlie the retraction phase. The currentsunderlying the BMPs were seen at a variety of voltages,allowing us to determine the voltage dependence of the cur-rents underlying the retraction phase. Currents recorded intraces showing a BMP were subtracted from currents measuredin the same cell, at the same voltage, in the absence of a BMP(BMPs were prevented by treating ganglia with tetrodotoxin),allowing us to estimate the currents that are exclusively attrib-

FIG. 3. CBI-2 elicited EPSPs are not detectedduring retraction phase of CBI-2-generated BMP. A:in protraction-phase neurons B34, B63, and B31firing of CBI-2 at a fixed rate for 10 s elicitedfacilitating fast EPSPs that generate a biphasic singlecycle of BMP. By contrast, shorter trains of CBI-2firing during the retraction phase do not cause de-tectable EPSPs. B: a fast sweep of the 1st part of thetrain marked as B in A demonstrates a train offacilitated EPSPs that increase their amplitude from�1 to �5 mV in B34 and B63. C: in contrast, a trainof similar duration and rate failed to elicit detectableEPSPs during retraction phase. The amplitude ofEPSPs progressively increased during the repeatedtrains.

FIG. 4. The postsynaptic change in resistance during retraction does notblock depolarization in response to a current pulse. Injection of 2 depolarizingcurrent pulses into B34 (A) and B63 (B) during and following the retractionphase demonstrates changes in resistance. During retraction, B31 shows thesame hyperpolarization seen in B34 and B63. the input resistance of B34 andB63 both were decreased by �50% during the retraction phase.



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utable to the presence of a BMP. The current-voltage relation-ship for the retraction phase was then plotted, and a linearregression best fit was calculated. The best fit linear regressionshowed that the currents underlying the retraction phase havea net reversal potential of �61 mV, and the change in inputresistance caused by the retraction phase is 1.01 M� (Fig. 5B).The input resistance of B31/B32 at rest was calculated fromconductances recorded in response to voltage steps in B31/B32at voltages in which the current-voltage relationship is ohmic.The measured passive input resistance was 2.71 M�. The1.01-M� change in input resistance during the retraction phasein B31/B32 would have reduced the input resistance to 1.70,which is a 37.25% reduction in the input resistance. Thuscurrent adequate to cause a 20-mV voltage change at restwould cause a 13-mV voltage change during the retractionphase of a buccal motor program. These data are consistentwith those in Fig. 4 and indicate that the shunting of theCBI-2-induced EPSP by the increase in conductance duringretraction would be adequate to reduce the amplitude of anEPSP from CBI-2 but would be too small to block completelythe CBI-2-induced EPSP. Thus another mechanism must ac-count for the complete block of the EPSP.

Complete block of CBI-2-elicited EPSPs is related to firingof B64

A number of retraction-phase interneurons have been iden-tified (Hurwitz and Susswein 1996; Nargeot et al. 1999; Plum-mer and Kirk 1990). The most prominent of these, B64, issufficient to induce the retraction phase (Hurwitz and Susswein1996). In spontaneous or elicited BMPs, a burst of spikes inB64 signals the start of the retraction phase. In addition, if B64is stimulated and begins to fire prematurely this firing leads tothe termination of protraction and an immediate, prematureonset of the retraction phase (Hurwitz and Susswein 1996).Because firing in B64 is a fixed marker of the start of retrac-

tion, we examined whether the firing of B64 is temporallyrelated to the block of the CBI-2-elicited EPSPs in protraction-phase neurons.

During CBI-2-initiated BMPs, the termination of the pro-traction phase was correlated with firing in B64 as well as withthe block of CBI-2-elicited EPSPs in the protraction-phaseneurons (Fig. 6A). In one experiment, stimulating B64 to fireduring the protraction phase caused a premature termination ofthe protraction phase (Fig. 6B) as was described previously(Hurwitz and Susswein 1996). This stimulus also caused apremature initiation of the retraction phase (see the 1st and the2nd cycles of BMP in Fig. 6B). Furthermore, firing B64 duringthe protraction, before it would have been activated withoutexperimenter intervention, led to three cycles of BMP in therecorded period instead of two (CBI-2 firing was maintainedfor 50 s, compare Fig. 6, A and B). In addition, the prematureonset of firing in B64 also blocked the CBI-2-elicited EPSPs inthe protraction-phase neurons (n � 7). This experiment indi-cates that firing of B64 causes the block of the CBI-2-elicitedEPSPs in protraction-phase neurons in addition to initiating theretraction phase.

The block of CBI-2-initiated EPSPs caused by firing B64 couldbe mediated via direct effects of B64, or via indirect effects, byB64 recruiting other retraction-phase neurons, which in turn couldblock the EPSPs. If the effect of firing B64 was via interveningneurons, the block of CBI-2-induced EPSPs should be eliminated,and the EPSPs should be restored when the buccal ganglia arebathed in HiDi saline (see METHODS), which elevates the firingthreshold of neurons and therefore makes it more difficult torecruit potential followers of B64.

To test whether the inhibitory effects of B64 stimulationmay be mediated via intervening neurons, various protraction-phase neurons were examined during the combined firing ofB64 and CBI-2, or during alternated firing of B64 and CBI-2,while both the buccal and cerebral ganglia were bathed in HiDisaline. To increase the size of the EPSPs, the various protrac-

FIG. 5. Change in membrane resistance during the retrac-tion phase in B31/B32. A: difference currents at the transitionfrom protraction to retraction during spontaneous buccal motorprograms recorded at different voltages in a single B31/B32neuron. In this experiment, currents underlying spontaneousBMPs were recorded in voltage-clamp conditions at differentvoltages. The currents recorded were subtracted from thoseseen in the absence of a BMP at the same voltage, yielding thedifference currents: that is, currents that are attributed only tothe BMP. The vertical dashed line shows the point of transitionfrom protraction (which causes inward currents) to retraction(which causes outward currents). The horizontal dotted linemarks 0 current. B: a linear best fit line was drawn for all valuesof the peak outward current recorded at different voltages (22current measurements from 8 B31/B32 neurons). The graphshows that there is a 1.01-M� change is resistance during theretraction phase.



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tion-phase neurons were held at –80 mV via injection ofconstant hyperpolarizing current. To control the firing fre-quency of B64, the neuron was stimulated with brief depolar-izing pulses, which initiated individual spikes.

CBI-2-elicited EPSPs in protraction-phase neuron B34 wereblocked by firing B64 in the presence of HiDi saline only whenB64 and CBI-2 fired simultaneously (Fig. 7). Driving CBI-2with trains of spikes that are followed by a number of secondsof rest elicited trains of facilitating EPSPs in B34. During thefirst minute of stimulation, the facilitation of the EPSPs growsfrom burst to burst (not shown). After the first minute, facili-

tation from burst to burst is constant (Fig. 7A). Firing of B642 s before firing CBI-2 did not affect the CBI-2-elicited EPSPs(Fig. 7B). By contrast, firing of B64 and CBI-2 simultaneouslycompletely blocked CBI-2-elicited EPSPs (Fig. 7C). This ex-periment indicates that B64 probably inhibits CBI-2-elicitedEPSPs directly because the inhibition is seen in HiDi saline. Italso suggests that B64 may produce presynaptic inhibition ofthe CBI-2 processes because the block is critically dependenton the simultaneous activity of CBI-2 and B64. Data similar tothose in Fig. 6 were also obtained when recordings were madefrom additional protraction-phase neurons, such as B31/B32,B61, and B63 (n � 11).

Block of CBI-2-induced EPSPs is caused bypresynaptic inhibition

A conclusive demonstration that B64 acts on CBI-2 processeswould require recordings from the CBI-2 processes within theneuropile of the buccal ganglia. Attempts to perform such record-ings were not successful in our hands (n � 11), (but see Sanchezand Kirk 2001). We therefore gathered indirect evidence support-ing the hypothesis that B64 directly inhibits CBI-2 neurites andthereby prevents them from releasing transmitter.

SIMULTANEOUS FIRING OF B64 AND CBI-2 BLOCKS FACILITATION

BUILD-UP OF CBI-2-ELICITED EPSPS. Previous data indicated thatthe facilitation of the EPSPs induced in protraction-phaseneurons by CBI-2 is a presynaptic process (Sanchez and Kirk2000). This facilitation is strongly affected by small changes inthe firing frequency of CBI-2 (Hurwitz et al. 2003). If the blockof the EPSPs induced by CBI-2 arises via presynaptic inhibi-tion, which causes a complete block of transmitter release, thefacilitation should be affected by the block in a manner similarto that caused by a complete interruption of CBI-2 firing. Bycontrast, if the block of the EPSPs arises as a result of apostsynaptic mechanism, the facilitation should be unaffectedby the block.

To examine the effects of firing B64 on the facilitation,CBI-2 was stimulated in bursts so that the bursts of EPSPsrecorded in B34 reached a steady-state facilitation (n � 3).These experiments were performed in a HiDi solution, and B34was held at –80 mV. In these conditions, firing CBI-2 in trainsdrives repeated bursts of EPSPs with summated amplitudesthat reach �35 mV at the end of a burst (Fig. 8A).

On the background of trains of current pulses applied toCBI-2 alone, B64 and CBI-2 were stimulated in tandem fourtimes. The simultaneous stimulation of B64 and of CBI-2blocked the EPSPs in B34 (Fig. 8B). Following the four stimuliwith combined trains of stimuli to both CBI-2 and B64, firingof CBI-2 alone again elicited EPSPs in B34. However, theamplitude of these EPSPs was decrease with respect to thatseen prior to the firing of B64. The amplitude of the EPSPsreturned to the values seen before the B64 stimulation onlyafter three bursts in which CBI-2 was stimulated alone (18 safter the end of the B64 stimulus).

Skipping four trains of CBI-2 spikes produced effects thatwere similar to those produced by the simultaneous stimulationof B64 and CBI-2. When CBI-2 stimulation was resumed, theamplitude of the summated EPSPs was reduced with fullrecovery of summated EPSPs amplitudes being observed onlyby the third train after the pause (Fig. 8C). These results

FIG. 6. B64 firing is temporally related to block of the CBI-2 elicitedEPSPs. A: tonic firing of CBI-2 for 50 s generated 2 cycles of biphasic BMP.The protraction phase was monitored via extracellular recording from I2n andvia intracellular recording from B61. The retraction phase was monitored viathe Rn activity that outlast the I2n activity and via intracellular recording fromboth B64 neurons. The termination of the protraction phase was correlated withfiring in B64 as well as with the block of CBI-2-elicited EPSPs in theprotraction-phase neurons. B: prematurely firing a single B64 neuron initiateda plateau depolarization in both B64 neurons and also led to 3 cycles of BMPin the recorded period instead of 2. During the retraction phases induced bypremature firing of B64, there was a block of CBI-2-elicited EPSPs identicalto that seen when B64 is recruited without depolarizing it. 2 in A, thetermination of the retraction phase.2 in B, CBI-2-elicited EPSPs that followtermination of the retraction phase.



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indicate that the simultaneous firing of B64 and CBI-2 affectsthe decay of facilitation in a manner similar to that caused bya complete lack of CBI-2 stimulation.

DISSOCIATION OF INHIBITION AND BLOCK OF EPSPS. B64 pro-duces postsynaptic inhibition of the protraction-phase neuronsas well as a block of the EPSPs from CBI-2. The data abovesuggest that these are separable processes. Additional evidencein support of this suggestion could be obtained if a secondneuron could be found that causes postsynaptic inhibition ofthe protraction-phase neurons, similar to that caused by B64,but that does not also block the CBI-2-induced EPSPs.

We have found such a neuron, which was not previouslyidentified and which we now name B54 (see Fig. 1A). B54 hasa soma �150% larger than that of B64. It is located medial to

B64 (n � 9). We have not examined systematically the role ofB54 in CBI-2 generated BMPs. B54 is similar to B64 in itsability to hyperpolarize protraction-phase neurons, includingB34 (Fig. 9). In addition, the increase in the conductance ofprotraction-phase neurons elicited by firing B54 is similar tothat caused by B64 (not shown, n � 5). B54 can be distin-guished from B64 because a brief depolarization of B64 elicitsa plateau potential that long outlasts the stimulus (Hurwitz andSusswein 1996), whereas B54 does not display this property.By contrast to B64, firing in B54 did not block CBI-2-elicitedEPSP (Fig. 9C), although it did cause a 50% decrease in theamplitude of the EPSPs as would be predicted if it shunted theEPSPs via a conductance increase IPSPs. This inhibition canbe seen by comparing the EPSP amplitudes resulting fromcombined bursts of CBI-2 and B54 (Fig. 9C) to the EPSPsamplitudes resulting from driving CBI-2 alone (Fig. 9B). Thesedata are consistent with the interpretation that postsynapticinhibition is separable from the block of CBI-2 induced EPSPsand provide further evidence that the EPSP block arises frompresynaptic inhibition.

Because B54 appears to exert only postsynaptic actions,one would predict that the simultaneous firing of CBI-2 andB54 would affect the size of summating EPSPs but not thefacilitation of the CBI-2-elicited EPSPs as does the simul-taneous firing of CBI-2 and B64. This prediction wasconfirmed in five experiments (Fig. 10). In the absence ofeither B54 or B64 stimulation, three trains of stimuli toCBI-2 elicited a gradual build-up in the amplitude of theEPSPs recorded in B34 (Fig. 10A). When CBI-2 and B54were stimulated together, the amplitude of the EPSPs elic-ited by stimulating CBI-2 was decreased (compare the 1stand 2nd trains in Figs. 10B to the 1st and 2nd trains in A).However, when B54 was not stimulated during the thirdtrain, the EPSPs elicited by CBI-2 were fully facilitated, asthey would have been if there had been no B54 stimulationin the previous trains (compare last trains of EPSPs in Fig.10, A and B). By contrast, when CBI-2 and B64 werestimulated in tandem during the first two trains, the CBI-2-elicited EPSPs were not seen, and in their place, the char-acteristic B64-elicited IPSPs were observed (Fig. 10C, 1st 2trains). When B64 was not stimulated during the third train,the EPSPs elicited by CBI-2 were expressed. However, inplace of full recovery of facilitation during the third train,like the one observed when B54 was not stimulated, skip-ping the third train of firing in B64 caused a reduction in theamplitude of CBI-2-elicited EPSPs with respect to theamplitude seen when B64 was not stimulated. Furthermore,during the third train of EPSPs, when B64 was not stimu-lated, the EPSPs elicited by CBI-2 were comparable to thoseseen during the first train of CBI-2 stimulation alone (com-pare the last train of EPSPs in Fig. 10C to the 1st train in A).Similar results were also obtained when another protraction-phase neuron, B63, was monitored in place of B34 (notshown, n � 2). These data indicate that the postsynapticinhibition of protraction-phase neurons by B54 and B64reduces the amplitude of the EPSPs elicited by CBI-2.However, only the presynaptic inhibition elicited by B64blocks synaptic release from CBI-2 terminals and therebyprevents the gradual buildup of EPSP facilitation.

FIG. 7. Combined, but not alternated firing of B64 and CBI-2 eliminatesCBI-2-elicited EPSPs. CBI-2 was stimulated at a rate of 10 trains/min, eachtrain with a 1-s duration at 16 Hz. This protocol leads to a progressive increasein the amplitude of the summated EPSPs over 1 or 2 min, until reachingsteady-state values. The figures shown are after the EPSPs in B34 have reachedsteady-state values. The bars indicate the maximum amplitudes of the sum-mated EPSP. B34 was hyperpolarized to approximately �80 mV throughoutthe experiment via constant current injection. High divalent (HiDi) saline wasreplacing artificial seawater (ASW). A: the EPSP produced in B34 by stimu-lating CBI-2 in the absence of activity in B64. B: when B64 was stimulatedseveral seconds preceding CBI-2 stimulation, CBI-2-elicited EPSPs wereunaffected. C: by contrast, when B64 fired simultaneously with CBI-2, theEPSPs were blocked. The recording of CBI-2 is also indicative of the timingsof CBI-2 activity in A and B.



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B64 CAN ALSO ELICIT PURELY PRESYNAPTIC EFFECTS. The dataabove provide strong evidence that the pre- and postsynapticinhibitions produced by B64 are separable processes, and theeffects of each can be characterized. Further evidence that B64can produce both pre- and postsynaptic inhibition could bemarshaled if an additional neuron was found that displayedonly presynaptic inhibition when B64 is fired with no concom-itant postsynaptic inhibition.

B8 was found to be such a neuron (n � 9). CBI-2 elicited aslow EPSP in B8. The slow EPSP was also present in HiDisaline, indicating that the connection is likely to be monosyn-aptic (Fig. 11A). In addition, firing of B64 did not inhibit B8 asit did the protraction-phase neurons but rather elicited a weakconductance decrease (Fig. 11B—compare the responses tocurrent injections just before and after firing of B64 with thoseduring B64 firing, which also elicits IPSPs in B61).

A conductance decrease should increase the amplitude of CBI-2-elicited slow EPSP in B8 if firing of CBI-2 precedes the firingin B64. Such an increase was seen when B64 was fired during the

slow EPSPs elicited by CBI-2 (Fig. 11C1). In this experiment, aseries of five CBI-2 bursts were elicited, and B64 was fired duringthe slow EPSPs elicited by the second and third burst. In theabsence of B64 firing, CBI-2 elicited six spikes. By contrast, whenB64 was also fired, CBI-2 elicited eight spikes. There is asignificant delay between firing of CBI-2 and the expression of theslow EPSP in B8, which permits the expression of B64 effects onthe ongoing EPSP (i.e., B64 was fired during the slow EPSPrather than during CBI-2 firing). However, when B64 was firedsimultaneously with CBI-2, it completely blocked the slow EPSPin B8 (Fig. 11C2). Thus firing CBI-2 alone evoked five to sixaction potentials in B8. When CBI-2 and B64 were fired together,no spikes were seen in B8 (compare the responses to the 1st andthe last trains of CBI-2 firing with that of the 2nd train). Thesedata conclusively illustrate that the presynaptic and postsynapticeffects of firing B64 are separable. Firing B64 in tandem withCBI-2 causes presynaptic inhibition, whereas firing of B64 intandem with the slow EPSP elicited by CBI-2 causes postsynapticexcitation.

FIG. 8. Effects of B64 activity are equivalentto those produced by rest in CBI-2. A: CBI-2was repetitively stimulated so those bursts ofEPSPs in B34 were fully potentiated. The barsindicate the maximum amplitudes of the sum-mated EPSP. B: when B64 and CBI-2 weresimultaneously activated during the 3rd, 4th,5th, and 6th bursts of stimulation, the EPSPs inB34 were eliminated completely. After the si-multaneous stimulation of CBI-2 and B64, the1st bursts of EPSPs recorded in B34 were de-creased in size, indicating a loss of facilitation.C: when CBI-2 stimulation was skipped for 4trains, 3 consecutive bursts of spikes in CBI-2were required until the amplitude of the facili-tated EPSPs was restored to the amplitude be-fore the interruption in CBI-2 stimulation. Therate at which EPSP amplitude was restored wassimilar when B64 was stimulated with CBI-2and when CBI-2 stimulation was interrupted.This experiment was performed in HiDi saline,and facilitation may differ from that seen inASW (Hurwitz et al. 2003; Sanchez and Kirk2001). B34 was held at �80 mV.

FIG. 9. B54 causes inhibitory postsynaptic potentials(IPSPs) in B34 that decrease the amplitude but do not blockCBI-2 elicited EPSPs. A: brief depolarization of B54-evokedspikes, which elicited a train of IPSPs that hyperpolarized B34by several milivolts. B: when CBI-2 spikes where triggeredduring firing of B54, they elicited facilitating EPSPs in B34.C: firing a train of spikes in CBI-2 in the absence of B54spikes evoked facilitating EPSPs in B34 that were larger inamplitude by 50%. This experiment was performed in HiDisaline while the neurons were held at their resting potential(approximately �60 mV).



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D I S C U S S I O N

Neural circuits in many animals are designed to transform atonic input into rhythmic output. Investigating the mechanismsunderlying the transformation of a tonic input into a rhythmicoutput in the Aplysia buccal motor system provides insight intothe general question of how to design a circuit to convert atonic input into a rhythmic output.

The data presented in this paper, in addition to those shownpreviously, indicate that at least three mechanisms contributein parallel to the transformation of a tonic input to CBI-2 intoa phasic motor output. First, the buccal CPG that is responsiblefor generating protraction-retraction sequences consists of sep-arate, mutually inhibitory protraction-phase and retraction-phase interneurons (Hurwitz and Susswein 1996; Hurwitz et al.1997). CBI-2 monosynaptically recruits the protraction-phaseneurons but not the retraction-phase neurons (Hurwitz et al.,2003). The protraction-phase neurons in turn recruit retraction-phase neurons, with a delay, via a presently unidentified

element that has tentatively been named the z cell (Baxter et al.1997). Activation of the retraction-phase neurons leads toinhibition of protraction-phase neurons (Hurwitz and Suss-wein 1996; Hurwitz et al. 1997). Thus CBI-2 inducesbiphasic activity by activating protraction, which itself thenactivates retraction and thereby shuts itself off. Second,phasic feedback from the buccal CPG to the cerebral gangliapostsynaptically inhibits CBI-2 (Hurwitz et al. 1999b;Rosen et al. 1991), leading to a rhythmic pattern of CBI-2firing (Rosen et al. 1991). CBI-2 will then fire and excite theprotraction-phase neurons in the buccal ganglia during onephase of a cycle. Constraining the firing of CBI-2 to theprotraction phase restricts the recruitment of buccal gan-glion protraction-phase neurons by CBI-2 to the appropriatephase of a motor program and inhibits the firing of CBI-2during the inappropriate phase. Last, we have now shownthat if the CBI-2 neuron is sufficiently excited and thereforefires during the retraction phase in spite of the inhibition

FIG. 10. Coactivation of CBI-2 and B64, but not of CBI-2and B54, blocks build-up of facilitation of CBI-2-elicitedEPSPs. A: 3 bursts of action potentials in CBI-2 elicited 3 trainsof monosynaptic EPSPs. The trains showed inter- and intra-train facilitation of both fast and slow EPSPs. B: coactivation ofCBI-2 and B54 for 2 bursts was followed by single train inwhich CBI-2 firing was triggered alone. Co-activation of B54and CBI-2 caused a decrease in EPSPs amplitude, but the 3rdtrain, in which the EPSPs were elicited with B54 stimulation,was the same as that seen when B54 was not fired in any of the3 bursts (compare the last burst in B to the last burst in A). C:by contrast, when B64 and CBI-2 are coactivated during the 1st2 trains, the last amplitude of the EPSPs elicited during the 3rdtrain, when CBI-2 alone is stimulated, is similar to that seenduring 1st train when CBI-2 alone is active (compare the lastEPSP of the last train in C to the last EPSP of the 1st train inA). The ganglia were bathed in HiDi saline and the membranepotential of the neurons was left at rest (�55 mV).



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seen during this period, such firing does not elicit excitationof protraction-phase neurons in the buccal ganglia. The lackof excitation is a result of presynaptic inhibition from themajor retraction element of the CPG, B64, onto the output ofCBI-2 within the buccal ganglia.

Evidence in favor of presynaptic inhibition

Our evidence that B64 blocks CBI-2 output via presynapticinhibition is indirect. Presynaptic inhibition is most directlydemonstrated via intracellular recordings from the presynapticterminals (e.g., Baxter and Bittner 1991) and via quantalanalysis (see Boyd and Martin 1956), which compares thequantal number and the quantal content of a synapse while it isinhibited to the values before inhibition and then shows that thequantal number, rather than quantal content, is reduced bypresynaptic inhibition (e.g., Boyd and Martin 1956). In oursystem, a quantal analysis would be unlikely to provide usefulinformation because the synapse from CBI-2 to protraction-phase neurons is completely blocked during the retractionrather than merely being reduced. When a synaptic connectionis completely absent, it would unrealistic to estimate changesin the quantum number or the quantal content. In place of aquantal analysis, we have marshaled a great deal of indirectsupport for the contention that B64 presynaptically inhibits theterminals of CBI-2 on protraction phase. The indirect measuresof presynaptic inhibition are similar to those used previously inother systems that display presynaptic inhibition (Dudel andKuffler 1961; Peng and Frank 1989). First, we have shown thatoverriding the inhibition of CBI-2 during retraction and caus-ing it to fire does not cause excitation of the protraction-phase

neurons during the retraction phase and therefore does notaffect the BMPs recorded in the buccal ganglia (Figs. 2 and 3).Although retraction is characterized by postsynaptic inhibitionof protraction-phase neurons, particularly by B64, the lack ofany excitation during retraction could not be explained bypostsynaptic inhibition of the protraction-phase neurons be-cause such inhibition would attenuate the amplitude of theEPSP caused by CBI-2 by 37% (Fig. 5) but would not elimi-nate it completely (Fig. 4) as was observed. In other systems,a reduction of a synaptic potential beyond that which can beexplained by postsynaptic inhibition has been used as evidenceof presynaptic inhibition (Frost et al. 2003). Second, we haveshown that the block of excitation from CBI-2 to protraction-phase neurons is correlated precisely with firing in B64 aswould be expected if the block was exerted via a phasicpresynaptic inhibition (Fig. 7). The need for a precisely timedinhibitory input immediately preceding the input that is inhib-ited is a widely seen feature of presynaptic inhibition that iscaused by directly gated receptors (for review, see MacDer-mott et al. 1999). Third, we have shown that block of excitationfrom CBI-2 to protraction-phase neurons causes changes insynaptic facilitation (a presynaptic process) identical to thoseseen when CBI-2 does not fire, whereas postsynaptic inhibitiondoes not affect synaptic facilitation in this synapse (Fig. 8).Modulation of facilitation has been widely used as a monitor ofpresynaptic inhibition (Baxter and Bittner 1991). Fourth, wehave identified a neuron, B54 (Figs. 1 and 9), that producespostsynaptic inhibition of the CBI-2-initiated EPSPs similar tothat produced by B64 but does not produce presynaptic inhi-bition, thereby demonstrating the separate effects of pre- and

FIG. 11. B64 blocks CBI-2 excitation of B8 butdoes not cause postsynaptic inhibition of B8. A:CBI-2 causes a slow EPSP in B8. B: B64 causes aconductance decrease in B8. The bar indicates thevoltage change caused by a depolarizing pulse in theabsence of B64 activity. C1: during repeated trainsof CBI-2 spikes, B64 was triggered to fire subse-quent to CBI-2 in phase with the excitation andfiring of B8. In response to B64 activity, the firing ofB8 increased, i.e., instead of 6 spikes, 8 spikes wereevoked. This reflected the decrease in conductanceinduced by B64, which amplified the effect of theCBI-2-induced slow EPSP. C2: by contrast to theamplification of the CBI-2-induced EPSP when B64fired after CBI-2, simultaneous firing of B64 andCBI-2 blocked the CBI-2-elicited EPSPs.



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postsynaptic inhibition of the connection from CBI-2 to pro-traction-phase neurons (Figs. 9 and 10). Last, we have foundthat for some neurons, B64 produces only a presynaptic inhi-bition of the EPSP from CBI-2 with no postsynaptic inhibitionat all (Fig. 11), further emphasizing that pre- and postsynapticinhibition are separable processes.

MECHANISM OF PRESYNAPTIC INHIBITION. Presynaptic inhibitioncan be caused via directly gated channels (for review, seeMacDermott et al. 1999) as well as via second-messenger-mediated channels (Fossier et al. 1994). A number of cellularmechanisms have been proposed to explain presynaptic inhi-bition. These mechanisms lead to a reduction in Ca2� entryinto the presynaptic terminal or a decrease in the efficacy ofCa2� in producing transmitter release. Presynaptic inhibition isoften associated with a reduction in the size of the presynapticaction potentials, which may be correlated with depolarizationof the presynaptic terminals (e.g., Pearson and Goodman 1981)or with hyperpolarization of the terminals (Dudel and Kuffler1961; Kretz et al. 1986b). An increase in conductance in theterminal, independent of whether it causes a depolarization orhyperpolarization, would shunt the currents in the terminal andthereby reduce the amplitude of presynaptic spikes (Baxter andBittner 1991) or perhaps block their invasion into the terminal.In addition, depolarization itself reduces the size of actionpotentials and also causes partial inactivation of the presynap-tic terminal (Burrows and Matheson 1994). Presynaptic inhi-bition may also be caused by an inhibition of the voltage-dependent Ca2� currents (Kretz et al. 1986b; Wu and Saggau1997) by which Ca2� for synaptic release enters the cell or bya direct inhibition of transmitter release (Parnas et al. 2000).

Presynaptic inhibition of the CBI-2 synaptic output by B64leads to a complete block of the EPSP rather than to itsreduction. Mechanisms that reduce the size of the presynapticaction potential, without eliminating it, could not account for acomplete block of synaptic transmission. Depolarization of theterminal large enough to inactivate it completely, and therebyblock spikes would probably also affect facilitation of thesynapse differently from that observed and are therefore un-likely to explain presynaptic inhibition in our system. BlockingCa�2 entry via the regulation of Ca�2 channels is likely to bemediated via second-messengers, the operation of which wouldbe too slow to account for the precise timing that is seen in theeffects of B64 on CBI-2-induced EPSPs and are therefore alsounlikely to operate in our system. The most likely hypothesis toexplain presynaptic inhibition in our system is block of spikeinvasion into the terminals.

TRANSMITTER IDENTIFICATION OF B64. Additional evidence thatB64 causes presynaptic inhibition of CBI-2 could be gatheredby showing that exogenous application of the B64 transmitterproduces inhibition of CBI-2 similar to that produced by firingB64. However, the B64 transmitter has not yet been identified.In Aplysia, histamine release by identified neurons has beenshown to cause presynaptic inhibition in both the cerebral(Chiel et al. 1988) and the abdominal ganglion (Kretz et al.1986a). A number of buccal ganglia neurons use histamine astheir transmitter (Evans et al. 1999). Studies that have localizedtransmitters to neurons in the buccal ganglia effectively elim-inate histamine, GABA, dopamine, serotonin, and nitric oxide(NO) as transmitters used by B64 (Diaz-Rios et al. 1999, 2002;Evans et al. 1999; Jacklet and Koh 2001; Kabotyansky et al.

1998). Although, glutamate and acetylcholine have been dem-onstrated to affect network properties of the feeding CPG inLymnaea (Straub et al. 2002), their ability to mimic B64 effectshas not been tested.

Function of presynaptic inhibition

Many neural systems are characterized by multiple, parallelmechanisms that act to achieve a common design feature,similar to the multiple, parallel mechanisms that act to trans-form a tonic input to CBI-2 into a rhythmic motor output.Finding this feature in the buccal motor system therefore is notsurprising. Nonetheless, the existence of parallel means toachieve the same goal raises the question of what the presenceof presynaptic inhibition contributes to a system that alreadyhas at least two additional methods of converting a tonic inputto CBI-2 into a rhythmic motor output.

DIFFERENTIAL REGULATION OF CBI-2 EFFECTS IN DIFFERENT GAN-

GLIA. In the stomatogastic nervous system of the crab, asingle modulatory interneuron projects to two different gan-glia. In one, its terminal are presynaptically inhibited, leadingto phasic modulation, whereas in the other ganglion tonicmodulation is seen (Coleman and Nusbaum 1994). Presynapticinhibition of CBI-2 by B64 could also cause different patternsof activity in the cerebral and buccal ganglia of Aplysia. B64neurites are restricted to the buccal ganglia (Hurwitz andSusswein 1996), thereby limiting presynaptic inhibition ofCBI-2 to these ganglia. However, firing CBI-2 excites neuronsin both the cerebral (Morgan et al. 2002; Rosen et al. 1991) andthe buccal ganglia (Hurwitz et al. 2003; Jing and Weiss 2001;Sanchez and Kirk 2000). Excitation of CBI-2 that causes firingduring retraction could therefore drive CBI-2 followers in thecerebral ganglion, whereas CBI-2 followers in the buccalganglion are blocked. Depolarization of CBI-2 has been shownto cause firing in other CBIs (Rosen et al. 1991). Because manyof these CBIs fire during both protraction and retraction,excitation from CBI-2 during retraction would not interferewith the patterning and timing of firing of these neurons.

DIFFERENTIAL CONTROL OF FACILITATION. A second possiblefunction of the presynaptic inhibition produced by B64 may beto regulate the strong facilitation of the synapses from CBI-2 tothe protraction-phase neurons. Previous data (Hurwitz et al.2003; Sanchez and Kirk 2000), as well as data in this paper(Figs. 7 and 8), have shown that these synapses undergofrequency-dependent changes in amplitude, leading to a largefacilitation. The facilitation in the CBI-2-induced EPSP is acentral feature in the ability of CBI-2 to induce a buccal motorprogram because the amplitude of the EPSPs preceding theprotraction phase may grow from 0 to �10 mV (Hurwitz et al.2003). A decrease in the facilitation may significantly delay orblock a BMP (Hurwitz et al. 2003). In addition, small changesin the background firing frequency of CBI-2 can profoundlyaffect the amplitude of the PSPs during a subsequent burst ofactivity in CBI-2 (Hurwitz et al. 2003). As in most systems(Katz and Miledi 1967), facilitation of the CBI-2 to protractionneuron synapses is a presynaptic process (Sanchez and Kirk2000). Presynaptic inhibition assures that the possible occa-sional firing of CBI-2 during the retraction phase will not causean undesired modification in the amplitude of the PSPs duringthe subsequent protraction phase. Our data support the possi-



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bility that presynaptic inhibition has this function because theeffect of presynaptic inhibition on facilitation is equivalent tothat of a complete cessation of CBI-2 firing, whereas postsyn-aptic inhibition does not affect facilitation (Fig. 9).

REGULATING PEPTIDE RELEASE. CBI-2 contains a number ofpeptide co-transmitters that can initiate or modulate buccal motorprograms (Morgan et al. 2000). The release of peptide co-trans-mitters, as well as their effects, is strongly affected by variationsin firing pattern and frequency (Vilim et al. 2000). The presyn-aptic inhibition of CBI-2 may contribute to achieving a function-ally appropriate release of peptide co-transmitters.

DEVELOPMENTAL CONSTRAINTS. Biological features can be ex-plained by ontogenic or phylogenic constraints of a system(Gould 2002). Presynaptic inhibition of CBI-2 by B64 couldalso be explained in this way. B64 postsynaptically inhibitsprotraction-phase neurons in the buccal ganglia, while excitingretraction-phase neurons (Hurwitz and Susswein 1997). Duringdevelopment, appropriate synapses must be made between B64and the protraction- and retraction-phase neurons. BecauseCBI-2 fires during protraction, its neurites within the buccalganglia may share molecular markers with protraction-phaseneurons. Such markers may lead to the development of inhib-itory connections from B64.

Comparison to other systems

In most systems, presynaptic inhibition reduces the ampli-tude of synaptic potentials rather than blocking them com-pletely. By contrast, B64 blocks the synaptic output. Presyn-aptic block of a synapse is an effective mechanism for pre-venting a synapse from functioning at an inappropriate time orto an inappropriate stimulus. In the crayfish lateral giantinitiated tail flip, presynaptic inhibition blocks the sensoryinputs that initiate the response, so that a tail flip will not itselfinitiate a second tail flip, leading to habituation of the response(Krasne and Bryan 1973). Presynaptic inhibition also phasi-cally blocks the output of crab neuron MCN1 in the stomato-gastic ganglion but not in the commissural ganglion, therebycreating phasic firing in one ganglion, and tonic firing in theother. In addition, MCN1 activity initiates both the gastric milland pyloric rhythms. However, MCN1 is presynaptically in-hibited during one phase of the gastric mill rhythm, therebydecreasing the recruitment of the pyloric rhythm. Thus presyn-aptic inhibition participates in the coordination of two rhythmicbehaviors (Bartos and Nusbaum 1997)

Cerebral to buccal interneurons similar to the CBIs inAplysia have been described in a number of different gastro-pods (Elliott and Susswein 2002). Similar to the CBIs, theseneurons also receive tonic input and act on a buccal ganglionCPG that generates a phasic response. Both tonically andphasically firing neurons have been identified in Pleurobran-chaea (Gillette et al. 1982; Kovac et al. 1982, 1983a,b 1986) aswell as in Limax (Delaney and Gelperin 1990a,b). In Lymnaea,a single phasically firing CBI-like neuron has been identified(McCrohan and Kyriakides 1989). The possibility that presyn-aptic inhibition contributes to the conversion of a tonic input toa phasic output in these systems has not been examined.

G R A N T S

This work was supported by National Institute of Mental Health GrantsMH-35564 and MH-50235, by Human Frontier Science Program Grant LT-

0464/1997, and by grants 413/97 and 357/02–17.2 from the Israel ScienceFoundation. We thank J. Koester for help in performing some experiments.

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