Article history:Accepted 11 June 2008Available online 20 June 2008
Electrical synapses play an important role in signaling between neurons and the synapticconnections between many neurons possess both electrical and chemical components.Although modulation of electrical synapses is frequently observed, the cellular processesthat mediate such changes have not been studied as thoroughly as plasticity in chemicalsynapses. In the leech (Hirudo sp), the competitive AMPA receptor antagonist CNQX inhibitedtransmission at the rectifying electrical synapse of a mixed glutamatergic/electricalsynaptic connection. This CNQX-mediated inhibition of the electrical synapse wasblocked by concanavalin A (Con A) and dynamin inhibitory peptide (DIP), both of whichare known to inhibit endocytosis of neurotransmitter receptors. CNQX-mediated inhibitionwas also blocked by pep2-SVKI (SVKI), a synthetic peptide that prevents internalization ofAMPA-type glutamate receptor. AMPA itself also inhibited electrical synaptic transmissionand this AMPA-mediated inhibition was partially blocked by Con A, DIP and SVKI. Lowfrequency stimulation induced long-term depression (LTD) in both the electrical andglutamatergic components of these synapses and this LTDwas blocked by SVKI. GYKI 52466,a selective non-competitive antagonist of AMPA receptors, did not affect the electrical EPSP,although it did block the glutamatergic component of these synapses. CNQX did not affectnon-rectifying electrical synapses in two different pairs of neurons. These results suggest aninteraction between AMPA-type glutamate receptors and the gap junction proteins thatmediate electrical synaptic transmission. This putative interaction between glutamatereceptors and gap junction proteins represents a novel mechanism for regulating thestrength of synaptic transmission.
Electrical synapses provide a direct pathway for ionic andbiochemical communication between cells and play a critical
rrell).droxy-5-Methyl-4-Isoxazoavalin A; DIP, Dynamin In
er B.V. All rights reserved
role in neuronal signaling. In neural networks which containlarge numbers of cells, such as in cortical circuits or the retina,electrical synapses play a critical role in synchronizingactivity between interconnected neurons (Roerig and Feller,
le Propionic Acid; CNQX, 6-Cyano-7-nitroquinoxaline-2,3-dione;hibitory Peptide; EPSP, Excitatory Postsynaptic Potential; LTD, Long
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2000; Bennett and Zukin, 2004; Meier and Dermietzel, 2006).Many synapses possess both electrical and chemical compo-nents and it is now apparent that plasticity in the chemicalsynaptic component can lead to changes in the electricalcomponent (Johnson et al., 1994; Smith and Pereda, 2003;Pereda et al., 2004). In addition, a number of recent studieshave shown that gap junctions are closely associated withpostsynaptic densities (PSD) (Sotelo and Korn, 1978; Rashet al., 2000; Lynn et al., 2001; Zoidl et al., 2007). This proximityof the electrical and chemical synaptic components mayallow for an interaction between the two modes of transmis-sion either by direct protein–protein interaction or by shortintracellular signaling pathways.
The leech (Hirudo sp) provides a useful system to studythe interaction between electrical and chemical synaptictransmission. The leech CNS is well characterized (seereview by Kristan et al., 2005) and there are a numbersynapses between readily-identifiable cells known to haveboth electrical and chemical components. For example, themechanosensory touch cells (T cells), of which there arethree bilateral pairs, form synaptic connections with eachother that have both an electrical and chemical component(Nicholls and Baylor, 1968; Baylor and Nicholls, 1969). In thisstudy, the relationship between electrical and glutamatergictransmission in the T-to-T synaptic connection was inves-tigated. Electrical synaptic transmission was found to beinhibited by both CNQX (a competitive antagonist of AMPA/Kainate glutamate receptors) and AMPA (a selective agonistof AMPA/Kainate receptors). The CNQX/AMPA-mediatedinhibition can be at least partially blocked by concanavalinA (Con A) or dynamin inhibitory peptide (DIP), which inhibitsclathrin-dependent endocytosis, and by pep2-SVKI, a syn-thetic peptide that inhibits internalization of AMPA-typeglutamate receptors. These results indicate an interactionbetween glutamate receptors and the gap junction proteinsthat mediate electrical synaptic transmission.
2. Results
2.1. Properties of the T-to-T electrical synapse
As first described by Baylor and Nicholls (1969), the T-to-Tsynapse has a monosynaptic electrical component and apolysynaptic chemical component (Fig. 1A). It is impossible todistinguish the electrical and chemical components of the T-to-T synapse in normal saline at room temperature (Fig. 1B,top). However, the electrical EPSP can be isolated by recordingin 15 mM Mg2+ saline solution to selectively block chemicalsynaptic transmission (Fig. 1B, top; Del Castillo and Katz, 1954;Baylor and Nicholls, 1969). To confirm that 15 mM Mg2+ salinesolution removes the entire chemical component of the T-to-TEPSP, the ganglionwas cooled to approximately 15 °C, delayingthe onset of the chemical EPSP just enough to allow theelectrical and chemical components to be distinguished(Fig. 1B, bottom; also see Nicholls and Purves, 1972). In theselow temperature recordings the later chemical componentwas completely abolished when 15 mM Mg2+ saline wasapplied, leaving only the earlier electrical component. Allsubsequent intracellular recordings were conducted at room
temperature in 15 mM Mg2+ saline to eliminate chemicalsynaptic transmission unless otherwise stated.
Normally, electrical synaptic function is studied by mea-suring the coupling coefficient between two neurons linked byelectrical synapses. However, no current flows from thepresynaptic to the postsynaptic cell in the T-to-T electricalsynapse unless an action potential is initiated in the pre-synaptic neuron (Figs. 1C, D; Baylor and Nicholls, 1969; Acklin,1988). The dependence of the T-to-T electrical connection onaction potential firing indicates that this electrical synapsehasa voltage threshold which shifts from a non-conducting to aconducting stateupon thearrival of anactionpotential, similarto what has been reported in giant motor synapses of thecrayfish (Furshpan and Potter, 1959). This property of T-to-Telectrical synapses is likely due to the voltage-sensitiveproperties of the gap junction proteins (innexins) thatmediateelectrical synaptic transmission between T cells and isconsistent with observations from vertebrate connexins thathave a relatively high voltage threshold (−30 to 0 mV; Werneret al., 1989; Ebihara et al., 1999; White et al., 1999; Rela et al.,2007). The latency of the EPSP to the action potential was verybrief, ≤0.6 ms and persisted in 15 mm Mg2+ saline, consistentwith an electrical synapse (Del Castillo and Katz, 1954).Sustained current flow between two T cells could be observedwhen a long, suprathreshold depolarizing current pulse wasinjected intooneTcell (Fig. 1D), indicating that thegap junctionsremain open for a time following the activating action potential.Hyperpolarizing current did not flow between the coupledT cells (Fig. 1C). All of these observations are consistent withearlier findings from Baylor and Nicholls (1969). Despite the factthat it was possible to measure sustained current flow betweenthe coupled T cells, most of the subsequent experiments exa-mining changes in the strength of the T-to-T electrical connec-tion weremade by measuring the electrical EPSP elicited by theTcell actionpotential. Thiswasdonebecause theelectrical EPSPis the physiologically-relevant function of the T-to-T electricalsynapse.
2.2. Effect of CNQX on the T-to-T electrical synapse
When the T-to-T synapse was recorded with 20 μM CNQX innormal saline, the EPSP amplitude decreased (Fig. 2A) indicat-ing that the chemical component of this synaptic connectionwas glutamatergic. As part of a control experiment, the effectof CNQX on the T-to-T synapse was tested in the presence of15 mMMg2+ saline, when only the electrical synapse would beactive (Del Castillo and Katz, 1954; Baylor and Nicholls, 1969).Surprisingly, the T-to-T electrical EPSP was significantlyreduced in CNQX-treated ganglia when compared to a controlgroup in which CNQX was omitted (Fig. 2B two-way ANOVAtreatment effect F1, 36=36.8, Pb0.0001, time effect F3, 36=11.2,Pb0.0001, interaction effect F3, 36=4.3, Pb0.05. Newman–Keulspost hoc test showed significant differences at both 5 min and15 min recordings, Pb0.05). No changes in input resistancewere observed in either the CNQX-treated or saline controlgroups making it unlikely that the CNQX effect was due toshunting of current out of the cell (Fig. 2B bar graph one-wayANOVA PN0.05). Inhibition of the T-to-T electrical synapseby CNQX was concentration-dependent (Figs. 2C, D) withsignificant inhibition observed at 10, 20 and 200 μM CNQX
Fig. 1 – Properties of the T-to-T synapse. (A) The T-to-T synapse has both a polysynaptic chemical component and amonosynaptic electrical component. (B) 15 mM Mg2+ saline solution selectively blocks chemical synaptic transmission,revealing the electrical component of the T-to-T EPSP. The ganglion was cooled to 15 °C so that the early electrical componentand the later chemical component could be distinguished. The later component was completely abolished when 15 mM Mg2+
was applied, leaving only the electrical component. (C) Subthreshold depolarizing current (top pair of traces) and hyperpolarizingcurrent (bottom pair of traces) injected into the presynaptic T cell did not spread to the postsynaptic T cell. (D) Traces comparingthe response of the postsynaptic T cell to a brief (5 ms), suprathreshold current versus a long (250 ms), suprathreshold currentinjection to the presynaptic T cell (black and gray traces, respectively). The brief current injection produced a transient electricalEPSP that rapidly decayed back to the resting potential, however sustained current flow between the two T cells could beobserved during the 250 ms current pulse if an action potential was elicited in the presynaptic T cell (compare these traces tothose in panel C).
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(Figs. 2C, D one-way ANOVA Pb0.0001). GYKI 52466 (GYKI;20 μM), which is a non-competitive antagonist of AMPAreceptors (Donevan and Rogawski, 1993; Zappala et al., 2001),also blocked the chemical component of the T-to-T synapsewhen the EPSP was recorded in normal saline, just like CNQX,but had no effect on the electrical EPSP recorded in 15mMMg2+
saline (data not shown, comparedwith control group two-wayANOVA PN0.05).
How might CNQX mediate depression of the electricalsynapse? In addition to being a competitive antagonist, CNQXcan induce internalization of AMPA-type glutamate receptors(Lin et al., 2000). It was possible that there was an interaction
Fig. 2 – CNQX inhibited T-to-T electrical synaptic transmission and the effect of CNQX was concentration-dependent. (A)Representative traces in normal saline illustrating that the chemical component of the T-to-T synapse is inhibited by CNQXindicating that this synapse is glutamatergic. (B) In panels B–D, recordings were made in 15 mM Mg2+ to eliminate chemicalsynaptic transmission. 20 μM CNQX significantly reduced the T-to-T electrical EPSP (N=6). No change in input resistance wasobserved in either the CNQX-treated or saline control groups. (C) The inhibitory effect of CNQX increased in a concentration-dependent manner. No significant difference was observed between T cells treated with 2 μM CNQX compared to the controlgroup (N=3). 10 μM (N=5), 20 μM and 200 μM (N=5) CNQX significantly reduced the amplitude of the electrical EPSP with200μMbeing significantlymore effective than 10μMand 20μM. 10μMand 20μMCNQX did not show a significant difference.The superimposed curved line is a sigmoidal fit meant to illustrate the trend in the data. (D) Representative traces showing theeffect of increasing concentrations of CNQX on the T-to-T electrical synapse.
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between glutamate receptors and the gap junction proteins atthese synapses, such that AMPA receptors internalizationproduced a decrease in electrical synaptic transmission. Thereare no known antibodies that recognize leech versions ofglutamate receptorsor innexins, thereforeweexploredpotentialinvolvement of receptor internalization by applyingCNQX in thepresence of concanavalin A (Con A), which had been used toblock clathrin-dependent internalization of neurotransmitterreceptors (Berlin et al., 1978; Paatero et al., 1988; Mayer andVyklicky, 1989; Xiang et al 2002; Kim et al., 2004; Arttamangkul
et al., 2006). When Con A (600 μg/ml) was co-applied with CNQX,the CNQX-mediated depression of the electrical synapse wascompletely blocked (Fig. 3A, D two-way ANOVA between Con A+CNQXandCNQXgroup,Pb0.01). ConAapplicationby itselfhadno effect on the T-to-T electrical synapse.
Dynamin inhibitory peptide (DIP), a blocker of endocytosis(Marks and McMahon, 1998; Wigge and McMahon, 1998;Carroll et al., 1999; Morishita et al., 2005) was also tested. DIP(50 µM) was not as effective as Con A, but nevertheless didinterfere with CNQX-mediated depression of the electrical
Fig. 3 – Con A, DIP and SVKI blocked CNQX-mediated inhibition of T-to-T electrical transmission. (A) 600 μg/ml Con Acompletely blocked 20 μM CNQX-mediated inhibition of the T-to-T electrical synaptic transmission (N=5). (B) 50 μM DIPprevented the initial effect of 20 μMCNQX on the T-to-T electrical EPSP (N=5). The DIP+CNQX group did not significantly differfrom the control group at 5 min. At the end of 15 min, there are no significant difference between the DIP+CNQX group and theCNQX group. (C) 100 μM SVKI completely blocked 20 μM CNQX-mediated inhibition of T-to-T electrical synaptic transmission(N=5). (D) Representative traces from the 5 min post-treatment stage showing CNQX-mediated inhibition of the T-to-Telectrical synapse (top) and the ability of Con A and SVKI to block the effects of 20 μM CNQX (bottom).
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synapse. At the beginning of the CNQX treatment, DIP didblock depression of the electrical synapse (Fig. 3B, 5min). Two-way ANOVA showed three groups were significant differentfrom each other (Treatment effect F2, 40=18.0, Pb0.0001, timeeffect F3,40=9.1, Pb0.001, interaction effect F6,40=2.7, Pb0.05).The DIP+CNQX group was significantly different from theCNQX group at 5 min and 10 min (Newman–Keuls post hoc,Pb0.05). However, by the end of CNQX treatment there was nosignificant difference between the DIP+CNQX and CNQXgroups (Fig. 3B Newman–Keuls post hoc, PN0.05). The aboveexperiments were repeated using 100 µM DIP, but thisconcentration was no more effective than 50 µM DIP (datanot shown).
Pep2-SVKI (SVKI) is a synthetic peptide that blocks theinteraction between glutamate receptor subunits and PDZdomain-containing proteins such as glutamate receptor-interacting proteins (GRIPs) and protein interacting with Ckinase (PICK), prevented AMPA receptor internalization (Liet al., 1999; Daw et al., 2000; Thalhammer et al., 2002). To date,
SVKI has not been used on invertebrate neurons, butinvertebrate AMPA-type glutamate receptors do containPDZ-binding domains and the mechanisms involved inglutamate receptor trafficking appear to be conservedbetween vertebrates and invertebrates (Chang and Rongo,2005; Walker et al., 2006). Treatment of the postsynaptic T cellwith 100 μM SVKI completely blocked CNQX-mediated inhibi-tion of the T-to-T electrical synapse (Fig. 3C, D two-wayANOVA between CNQX+SVKI and control group PN0.1;between CNQX+SVKI and CNQX group Pb0.05). SVKI alonedid not affect electrical synaptic transmission. Taken together,the Con A, DIP and SVKI results support the hypothesis thatthe CNQX-mediated inhibition of the electrical synapseinvolves glutamate receptor internalization.
2.3. Effect of AMPA on the T-to-T electrical synapse
AMPA itself is also known to induce internalization of AMPA-type glutamate receptors and is more effective at stimulating
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internalization than CNQX (Man et al., 2000; Lin et al., 2000).Therefore, the effects of AMPA on T-to-T electricalsynaptic transmission were also examined. 100 μM AMPAcompletely abolished the T-to-T electrical EPSP by the end ofa 15 min treatment (Figs. 4A, B two-way ANOVA, treatmenteffect F2, 24=112.1 Pb0.0001; time effect F3, 24=58.7; Pb0.0001;interaction effect F6, 24=13.7 Pb0.0001). The effect of 10 μMAMPA was similar to 100 μM AMPA, but was slower todevelop and did not completely eliminate the electrical EPSP(Figs. 4A, B). Con A, DIP and SVKI were able to inhibit theinitial 10 µM AMPA-induced depression of the electricalsynapse, but could not prevent depression at later timepoints in the AMPA treatment (Fig. 4C two-way ANOVA,treatment effect F2,20=17.7 Pb0.001, time effect F3,20=17.7Pb0.001, interaction effect F6,20=3.7 Pb0.01; Newman–Keulspost hoc test 5 min Con A+AMPA group was significantlydifferent from AMPA group Pb0.01 but not significantlydifferent from control group PN0.1; Fig. 4D two-wayANOVA, F2,36=23.2 Pb0.0001, F3,36=8.2 Pb0.001, F6,36=3.64Pb0.01; Newman–Keuls post hoc test 5 min DIP+AMPAgroup was significantly different from AMPA group Pb0.05but not significantly different from control group PN0.05;Fig. 4E two-way ANOVA, treatment effect F=17.1 Pb0.001,interaction effect F6,20=3.1 Pb0.01; Newman–Keuls post hoctest 5 min AMPA+SVKI group was significantly differentfrom AMPA group Pb0.05 but not significantly differentfrom control group PN0.1). Neither concentration of AMPAhad a significant effect on input resistance, indicating thatAMPA's effect on the electrical EPSP was not due to ashunting of currents out of the cell (data not shown, one-way ANOVA PN0.05). This is not surprising given that AMPAreceptors rapidly desensitize. Again, these results are con-sistent with the hypothesis that there is an interactionbetween AMPA receptors and innexins at the T-to-T synapsein which ligand binding to the AMPA receptor inducesinhibition of the electrical synapse that involves AMPAreceptor internalization.
The effect of AMPA on chemical synaptic transmissionwas also tested by recording T-to-T synaptic transmission innormal saline since AMPA-induced internalization of AMPAreceptors would be expected to reduce the chemical/gluta-matergic component of this synapse. The decrease in the
Fig. 4 – AMPA inhibited T-to-T electrical synaptic transmissionBoth 10 and 100 μMAMPA significantly reduced the electrical EPSeach group). 100 μM AMPA abolished the T-to-T electrical EPSPslower to develop and did not completely inhibit the electrical EPstage showing the effect of 10 μM AMPA, (top traces) 100 μM AMCon A prevented the inhibitory effect of 10 μM AMPA on electrica15 min recordings in Con A+AMPA group still showed significanprevented the inhibitory effect of 10 μMAMPA on electrical transgroup (N=4). Again, DIP had no effect on AMPA-mediated inhibiblocked the inhibitory effect of 10 μM AMPA on electrical transmgroup, but had no effect on AMPA-mediated inhibition at later titransmission in normal saline indicating inhibition of both the cheMg2+ saline, Con A attenuated this effect. The total EPSP was inhiAMPA and ConA+AMPA group(10μMAMPA groupN=5; 10μMAwas shownbetween theAMPAand ConA+AMPAgroups at this teffect of 10 μM AMPA on T-to-T synaptic transmission at the 5 m
T-to-T EPSP during the first 5 min of 10 µM AMPA treatment innormal saline was substantially greater (≈80%) when com-pared to the level of inhibition during the same time point10 µM AMPA treatment in 15 mM Mg2+ saline (≈40%; Figs. 4A,F). This is consistent with both the chemical and electricalcomponents of the T-to-T synapse being inhibited by AMPAtreatment. Con A prevented the AMPA-mediated inhibition ofthe T-to-T synapse during the first 5 min of the AMPAtreatment in normal saline (Fig. 4F two-way ANOVA treat-ment effect F2,24 =32.5 Pb0.0001, time effect F3,24 =20.7Pb0.0001, interaction effect F6,24=5.9 Pb0.001; Newman–Keuls post hoc test 5 min Con A+AMPA group was signifi-cantly different from AMPA group Pb0.05 but not significantlydifferent from control group PN0.05), identical to the resultsobserved in AMPA+Con A experiments conducted in 15 mMMg2+ saline, but was unable to prevent AMPA-mediateddepression at later time points (Newman–Keuls post hoctest 15min Con A+AMPA groupwas not significantly differentfrom AMPA group PN0.1).
2.4. Effect of AMPA and CNQX on sustained current flowbetween T cells
The preceding experiments tested the effects of CNQX andAMPA on the transient electrical EPSP. To confirm that AMPAand CNQX were inhibiting electrical coupling, experimentswere conducted in which the sustained current flow betweenthe T cells was monitored. This was accomplished byapplying a 250 ms depolarizing current pulse to the pre-synaptic T cell that was sufficient to elicit a single actionpotential and then measuring the level of sustained depolar-ization in the postsynaptic T cell following the initial EPSP,approximately 200 ms from the beginning of the currentpulse (see Fig. 1D). A coupling coefficient was then calculatedusing the ratio of the postsynaptic membrane potential overthe presynaptic membrane potential. Care was taken to onlymeasure T-to-T current flow following a single actionpotential in the presynaptic cell since the current flowbetween the T cells may be affected by the number andfrequency of presynaptic action potentials. All recordingswere carried out in 15 mM Mg2+ saline and the sustainedcurrent flow between T cells was compared prior to and
and this effect was antagonized by Con A, DIP and SVKI. (A)P at each time point compared with the control group (N=5 inalmost completely while the inhibition of 10 μM AMPA wasSP. (B) Representative traces from the 5 min post-treatmentPA (middle traces) and 10 μM AMPA+Con A (bottom traces). (C)l transmission at the 5 min recording (N=4). Both 10 min andt inhibition compared with the control group. (D) 50 μM DIPmission at the first 5 min recording compared with the controltion at later time points. (E) 100 μM Pep2-SVKI completelyission at the first 5 min recording compared with the controlme points (N=4). (F) AMPA inhibited T-to-T synapticmical and electrical components of this synapse. As in 15mmbited at the end of the 15 min 10 μM AMPA treatment in bothMPA+ConAN=4; Con A groupN=4). No significant differenceime point. However, ConA effectively prevented the inhibitoryin recording compared with the control group.
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15 min after treatment with AMPA, CNQX or saline. 100 μMAMPA completely eliminated post-action potential currentflow between the T cells (Figs. 5A, D one-way ANOVA Pb0.05;Newman–Keuls post hoc AMPA-treated group significantlydifferent from the control group Pb0.05), a result identical tothe effect of this concentration of AMPA on the electricalEPSP. 200 μM CNQX also reduced the level of sustainedcurrent flow between the recorded T cells, although thiseffect was not statistically significant (Figs. 5B, D). The CNQX-treated coupling was not significantly different from the
control preparations tested, but was also not significantlydifferent from the AMPA-treated group (post hoc PN0.05).Earlier results indicate that CNQX is not as effective as AMPAin inhibiting the electrical EPSP (see Figs. 2 and 4) and it isalso likely that since the current injected into the presynapticT cell was minimal amount necessary to elicit a single actionpotential, that a “basement” effect for CNQX was observed.Nevertheless, these results show that treatments that inhibitthe electrical EPSP also reduce sustained current flowbetween the coupled T cells.
Fig. 5 – AMPA and CNQX inhibited the level of sustained current flow between T cells. (A) Traces showing the level of sustainedcurrent flow between two ipsilateral T cells prior to (black traces) and after (grey traces) a 15min application of 100μMAMPA. (B)Traces showing the level of sustained current flow between two ipsilateral T cells prior to (black traces) and after (grey traces) a15 min application of 200 μMCNQX. (C) Traces showing the level of sustained current flow between two ipsilateral T cells priorto (black traces) and after (grey traces) 15min in 15mMMg2+ saline (control experiments). (D) Bar graph summarizing the effectsof AMPA (N=3) and CNQX (N=5) on coupling between T cells.
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2.5. Modification of the T-to-T synapse during long termdepression
In order to examine whether decreases in the T-to-Telectrical EPSP has a functional role, low frequency stimula-tion (LFS) was used to induce long term depression (LTD) inthe T-to-T synapse. In all of the experiments made in normalsaline, which contains 1 mM Mg2+, it was impossible todistinguish the electrical and chemical components of theT-to-T EPSP. However in recordings by Baylor and Nicholls(1969), the chemical component of the EPSP was sufficientlydelayed so that the electrical component of the EPSP could bereadily observed. One difference between these earlierexperiments and our own recordings is that Baylor and
Nicholls (1969) used Mg2+-free saline. Recordings made inMg2+-free saline reproduced the findings of Baylor andNicholls (1969), showing a delay in the chemical componentof the EPSP that was not observed in recordings made in1 mM Mg2+ saline (Fig. 6A). There were no differences in thesize of the T-to-T EPSP recorded in 1 mM Mg2+ (2.6±0.1 mV)and those recorded in Mg2+-free saline (2.5±0.3 mV). It is notknown why eliminating Mg2+ delays the chemical EPSP. Onepossibility is that there is an increase in inhibitory synapticinput in the Mg2+-free saline, delaying the firing of theunknown neuron that mediates the polysynaptic EPSP.
In both normal saline and Mg2+-free saline, 900 stimuli LFS(1 Hz) induced identical levels of LTD in the T-to-T synapse(Fig. 6B, C normal saline group student t-test Pb0.001; 0 mM
Fig. 6 – The chemical and electrical components of the T-to-T synapse are depressed following low frequency stimulation-induced LTD. All the data are normalized to the baseline (EPSPpost training/EPSPpre training). (A) Representative traces showing thedifference between T-to-T EPSP elicited in 0 mM Mg2+ saline (top traces) versus normal saline with 1 mM Mg2+ (bottom trace).Note that in 0 mM Mg2+ saline, the chemical component of the EPSP is delayed just enough so that the electrical componentcan be observed. (B) Representative traces from pre-training recording (black trace) and post-training recording (gray trace).900 s LFS induce LTD in both chemical and electrical EPSP component of T-to-T synapse which can be totally blocked byPep2-SVKI. (C) Vertical bar graph illustrating that 900 s LFS (1 Hz) was able to induce long term depression of T-to-T synapse inboth normal saline group (N=4) and 0 mM Mg2+ group (N=4). 900 s LFS also induced a significant depression on Electrical EPSPin 0 mM Mg2+ group (N=4). (D) Vertical bar graph showing SVKI (N=4) prevented 900 s LFS-induced LTD relative to the controlgroup (N=5).
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Mg2+ group student t-test Pb0.01). Furthermore, in experi-ments carried out in Mg2+-free saline LTD, it was possible toobserve depression in both the electrical and chemicalcomponent of the EPSP (this later component is dominated
by the chemical EPSP, but electrical EPSP has not fullydecayed so it is referred to as the “total EPSP”). This LFS-induced LTD of the T-to-T synapse was blocked by SVKI(Fig. 6D electrical EPSP student t-test between 900 s LFS+SVKI
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group and 900 s LFS group, Pb0.001; student t-test betweenSVKI group and control groups, PN0.05) indicating a depen-dence for AMPA receptor internalization. No changes in inputresistance were observed in T cells that underwent LFS innormal saline (95.2±4.8% of initial input resistance), T cells in0 mM Mg2+ saline (93.0±1.7%) or the pep-SVKI-treated T cells(94.6±6.9%).
2.6. Effect of CNQX on the T-to-S electrical/chemicalsynapse
The effects of CNQX were examined on a second mixedglutamatergic/electrical synapse that between the T and Scells (Muller and Scott, 1981). The S cell is an interneuronthought to be critical for learning in the whole-body short-
Fig. 7 – CNQX reduces both the chemical and electrical componeshowing that 20μMCNQX reduced the electrical component andElectrical EPSP data were measured and normalized to the initialelectrical component of the T-to-S synapse while no change waselectrical synaptic transmission was observed after a 10minwascoupling prior to (top) and following (bottom) CNQX treatment. (D)(left) and L (right) cells was unaffected by a 15min application of 2membrane potential/postsynaptic membrane potential.
ening reflex (Sahley et al., 1994; Modney et al., 1997; Burrellet al., 2003; Burrell and Sahley, 2005). Action potentialselicited in a T cell produce a 1–2 mV, short latency electricalEPSP followed by a larger 4–6 mV chemical EPSP (Fig. 7B; alsosee Muller and Scott, 1981). There is a substantial delaybetween the start of the electrical EPSP and the start of thechemical EPSP; therefore, the two components can be readilydistinguished. CNQX completely blocked the chemical EPSP,indicating that this component of the T-to-S synapse wasglutamatergic, and also significantly reduced the T-to-Selectrical EPSP by approximately 40% (Fig. 7B two-wayANOVA, Treatment effect F1,20=30.6, Pb0.0001). These resultswere identical to the observed effects of CNQX at the T-to-Tsynapse. No change in input resistance in the postsynaptic Scells was observed between the control and CNQX groups.
nt of the leech T-to-S synapse. (A) Representative traceseliminated the chemical component of the T-to-S synapse. (B)level in 15 mM Mg2+ saline. 20 μM CNQX (N=6) inhibited theobserved in the control group (N=6). Partial recovery of T-to-Sh in normal saline solution. (C) Representative traces of R-to-RScatter plots showing that the coupling ratio between paired R0μMCNQX. Coupling ratio was calculated as the presynaptic
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2.7. Effect of CNQX on non-rectifying electrical synapses
The effect of CNQX was also tested on the non-rectifyingsynapse: between the paired Retzius (R) neurons (Fig. 7C;Stuart, 1970; Lent, 1977; De-Miguel et al., 2001; Garcia-Perezet al., 2004). In this electrical synapse, both positive andnegative currents flow equally well in both directions betweenthe coupled neurons. To examine the effect of CNQX onelectrical transmission between R cells, the coupling ratio wascalculated as the postsynapticmembrane potential/presynap-tic membrane potential and was tested prior to and followingCNQX treatment. As shown in Fig. 7C and D, the coupling ratiobetween paired R cells was not affected by a 15 min applica-tion of 20 μM CNQX (one-way ANOVA, PN0.05). Comparison ofCNQX-treated R cell pairs with initial recordings in normalsaline solution (Fig. 7C top trace) showed that CNQX did notinhibit non-rectifying electrical synapses (Fig. 7C bottomtrace). Another non-rectifying electrical synapse betweenpaired longitudinal motor neurons (L cells) was tested andthe coupling ratio was also not affected by CNQX treatment(data not shown, one-way ANOVA, PN0.05).
3. Discussion
In this study, we have shown that both CNQX and AMPAinhibited electrical synaptic transmission in the CNS of themedicinal leech. Both Con A and DIP, which inhibit endocy-tosis, blocked CNQX-mediated depression of the electricalsynapse and attenuated AMPA-mediated depression. SVKI, aninhibitor of AMPA-type receptor internalization, also blockedCNQX-induced depression of the electrical synapse andpartially blocked AMPA-mediated depression. SVKI alsoblocked LTD of the electrical and chemical components ofthe T-to-T synapse. GYKI 52466, a non-competitive glutamatereceptor antagonist, did not affect electrical synaptic trans-mission. CNQX-mediated depression of the electrical EPSPwasalso observed at the T-to-S synapse, but CNQXhadno effect oncoupling at the non-rectifying electrical synapses betweenpaired Retzius cells or paired longitudinal motor neurons.
AMPA receptors are constantly cycled between intracellu-lar stores and the cell surface (Malinow and Malenka, 2002;Sheng and Kim, 2002) and internalization of AMPA receptors isbelieved to be the major mechanism mediating long-termdepression (LTD) of glutamatergic synaptic transmission (Manet al., 2000; Lin et al., 2000; Kim et al., 2001). Previous studieshave demonstrated that CNQX and AMPA can induce inter-nalization of glutamate receptors in cultured hippocampalneurons with AMPA being much more effective than CNQX(Lin et al., 2000). It is thought that AMPA-mediated inter-nalization is initiated by two processes; one that depends oncurrent flux through the activated AMPA receptor channel anda second process that depends on direct protein-to-proteininteractions between the AMPA receptor and one or moreunknown molecules (Lin et al., 2000). The CNQX-mediatedinternalization of AMPA receptors is thought to be weaker, atleast in part, because CNQX can only activate the lattermechanism and does not elicit current flow through thereceptor. GYKI 52466 is not able to elicit AMPA receptorinternalization presumably because it is not a competitive
antagonist and therefore does not bind to the glutamate-binding site (Lin et al., 2000). Although CNQX/AMPA-mediatedinternalization of glutamate receptors has not been examinedin invertebrates, these receptors do undergo trafficking as aresult of LTP, serotonergic modulation and metabotropicglutamate receptor activation (Antonov et al., 2007; Chitwoodet al., 2001; Ji and Hawkins, 2003; Li et al., 2005; Grey andBurrell, 2008; Pan and Broadie, 2007). Furthermore, there isevidence of conservation in the cellular machinery used tomediate glutamate receptor trafficking between vertebratesand invertebrates (Dierkes et al., 1996; Chang and Rongo, 2005;Walker et al., 2006).
Our electrical synapse data parallel the AMPA/CNQX-mediated depression of glutamatergic synapses in a number ofinteresting ways. In both AMPA is much more effective thanCNQX and appears to depend on occupation of the glutamatereceptor binding site since GYKI 52466 had no effect. Thatinhibition of the electrical EPSP involved endocytosis is based onthe findings that Con A and DIP blocked CNQX-mediateddepression and partially disrupted AMPA-mediated depression.These results suggest an interaction between AMPA-type gluta-mate receptors and the gap junction proteins that mediateelectrical synaptic transmission. Specifically, CNQX- or AMPA-induced internalization of AMPA receptors may produce adecrease in electrical synaptic transmission due to an unidenti-fied linkage between the AMPA receptors and the gap junctionproteins. Such a mechanism is supported by the fact that SVKI,which specifically inhibits AMPA receptor internalization, blocksCNQX-mediated depression of the electrical synapse, attenuatesAMPA-mediated depression and blocks LTD of both the chemicaland electrical components of the T-to-T synapse. This ability ofSVKI to block LTD in the leech is consistent with some forms ofLTD observed in the vertebrate brain which is thought to beexpressed via the internalization of glutamate receptors (Kimet al., 2001). It is not knownat this timewhether this LTD requiresactivationofNMDAreceptors, butNMDAreceptor-dependentLTPand LTD have been observed at other synapses in the leech CNS(Burrell and Sahley, 2004; Li and Burrell, 2007).
Howmight internalization of glutamate receptors lead to adecrease in electrical synaptic transmission? One possibilityis that the gap junction proteins (innexins in the leech andother protostomal invertebrates) are co-internalized alongwith the glutamate receptors. Increasingly, molecular andelectrophysiological evidence supports the idea that there canbe an intimate association between the chemical andelectrical components of a synapse. Axosomatic and axoden-dritic gap junctions have been found to cluster at active zonesopposed to postsynaptic densities (PSDs), suggesting thatmembrane receptors and gap junction proteins can interactwith each other either by short-range intercellular signalingor by direct protein–protein interactions (Sotelo and Korn,1978; Rash et al., 2000; Lynn et al., 2001; Zoidl et al., 2007).Alternatively, depression of the electrical EPSP may notinvolve trafficking of the gap junction proteins themselves,but instead involve inhibition of gap junction function, e.g. byreducing current flow through the gap junctions. Unfortu-nately, it is impossible to directly monitor the trafficking ofeither innexins or glutamate receptors in the leech CNS sincethere are no known antibodies that recognize the leechversion of these proteins.
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Although CNQX-mediated inhibition of the electrical EPSPappears to be mediated by a purely endocytosis-dependentprocess (based on the effects of Con A, DIP and SVKI), AMPA-mediated inhibition of the electrical synapse was onlypartially blocked by drugs that blocked endocytosis. Thissuggests that AMPA application inhibited the electricalsynapse via two distinct mechanisms, one linked to inter-nalization of the AMPA-type glutamate receptors and one ormore additional processes that are independent of receptortrafficking. Examples of these latter processes includeincreased intracellular Ca2+, which is known to inhibitelectrical coupling (Connors and Long, 2004), or activation ofother modulatory neurons in the leech brain as a result ofAMPA application. Previous work has shown that serotonininhibits electrical synapses in the leech CNS (Colombaioni andBrunelli, 1988; Beck et al., 2002; Moss et al., 2005) and neuronalgap junctions have been shown to be modified by a varietymodulatory transmitters or hormones including serotonin,ecdysone, dopamine and acetylcholine (Piccolino et al., 1984;Teranishi et al., 1983; Moreno et al., 1991; Johnson et al., 1993,1994; Rorig and Sutor, 1996; Velazquez et al., 1997; Teshibaet al., 2001; Antonsen and Edwards, 2007).
It is also possible that the CNQX/AMPA induces depressionof the electrical EPSP by directly binding to the innexins orsome other protein that then acts on the innexins. Thisexplanation cannot be excluded given that no direct observa-tion of glutamate receptor or innexin trafficking could bemade. However, there have been no other reports of eitherCNQX or AMPA directly acting on either vertebrate orinvertebrate gap junction proteins and these drugs had noeffect on the non-rectifying electrical synapses between thepaired Retzius and longitudinal motor neurons. Furthermore,the ability of SVKI to block CNQX-mediated depression andattenuate AMPA-mediated depression suggests a direct role bythe glutamate receptors.
CNQX and AMPA did not inhibit electrical couplingbetween the paired Retzius interneurons and paired long-itudinal motor neurons. As in most invertebrates, gapjunctions in the leech are composed of innexin proteinswhich are encoded by a multi-gene family with 12 innexingenes (Hm-inx) currently identified in the medicinal leech(Dykes et al., 2004; Dykes and Macagno, 2006). Three types ofgap junction proteins are observed in the animal kingdom;connexins and pannexins in the deuterostomes (e.g. verte-brates and invertebrate chordates) and innexins in theprotostomal invertebrates, (e.g. arthropods, annelids andmollusks; Barbe et al., 2006). As with vertebrate electricalsynapses, innexin subtypes determine the functional proper-ties of the leech gap junction, such as whether current flowsequally well in both directions between the coupled neurons(non-rectifying electrical synapse) or exhibit a clear direction-ality of current flow (rectifying electrical synapse) andwhether both positive and negative current can pass throughthe gap junctions. It is possible that the inability of CNQX orAMPA to inhibit coupling between the Retzius or longitudinalmotor neurons may be due to a different compliment ofinnexin subtypes that make up these connections. Anotherpossibility is that the R-to-R and L-to-L gap junctions may notbe in close enough proximity to the glutamate receptors to beaffected by the CNQX/AMPA treatments.
AMPA receptor internalization plays a critical role in someforms of long-term depression (LTD; Luscher et al., 1999;Seifert et al., 2000) and a linkage between this process anddepression of the electrical EPSP would not be surprising insynapses that have both a glutamatergic chemical andelectrical component. If the glutamatergic component of asynapse is altered then it would be advantageous for theelectrical component to change in a parallel manner. Theresults presented here suggest that a linkage between theglutamatergic and electrical synaptic components does existand it is not the first evidence an interaction. LTP of theglutamatergic component of Mauthner cell synapses isaccompanied by potentiation of the electrical component(Smith and Pereda, 2003) and this potentiation of the electricalEPSP requires the activation of nearby chemically receptivezone(s) in the same synapse. This coordinated modulation ofboth electrical and glutamatergic synaptic transmission mayplay an important role in reshaping neural pathways for anumber of processes including sensory processing, learningandmemory, neural development and homeostatic processes.AMPA-mediated depression of the glutamatergic and electri-cal synapses may also provide a protective mechanismagainst glutamate-induced excitotoxicity.
4. Experimental procedures
Leeches, weighing 3 g, were obtained from a commercialsupplier (Leeches USA,Westbury, N.Y.) and kept in pondwater[0.52 g/L H2O Hirudo salt (Leeches USA Ltd.)] at 18 °C.Individual ganglia were dissected from the animal and placedin a recording chamber (1.5 mL) with constant perfusion(~2 ml/min). The dissections and recordings were carried outin leech saline containing: 115 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2,and 10 HEPES. In high Mg2+ saline experiments, 15 mM MgCl2was used, which replaced NaCl mole for mole.
Dual intracellular recordings were made by impalingneurons with intracellular glass microelectrodes using amicropositioner (Model 1480; Siskiyou Inc., Grants Pass, OR).Electrodes were pulled from borosilicate capillary tubing(1.0 mm outer diameter, 0.75 mm inner diameter; FHCBowdoinham, ME) to a resistance of 25–35 MΩ (SutterInstruments P-97; Novato, CA) and filled with 3 M potassiumacetate. Current pulses were delivered to the neurons using atwo-channel stimulator with stimulus isolation units (S88and SIU5, respectively; Astromed-Grass, West Warwick, RI).Signals were amplified with a bridge amplifier (BA-1S; NPI,Tamm, Germany) and then digitally converted (Digidata1322A A/D converter) for viewing and subsequent analysis(Axoscope 10; Molecular Devices, Sunnyvale, CA). Individualneurons were identified based on their position, size andaction potential shape. EPSPs were elicited by current injec-tions into the presynaptic cells at regular (5 min) intervals.The resting membrane potential of the postsynaptic neuron(T cell or S cell) was approximately −40 mV and washyperpolarized to −50 mV to prevent the initiation of actionpotentials. Since a decrease in input resistance can cause anapparent decrease in synaptic signaling, input resistance wasmeasured throughout each experiment by injecting negativecurrent pulse (0.5 nA, 500 ms).
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In temperature control experiments, a Peltier device and atemperature control system (HCPPS; ALA Scientific, Westbury,NY and TC-10; NPI, Tamm, Germany) were used to pre-coolthe perfusion solution just before it entered the recordingchamber. A thermal probe was placed in the recordingchamber to monitor the solution temperature in the recordingchamber. These low temperature recordings were used todelay the chemical EPSP in the T-to-T synapses to permitmeasurement of the electrical EPSP in normal saline.
For T-to-T experiments, synaptic transmission betweenipsilateral T cellswas tested every 5min. To isolate the electricalcomponent of the T-to-T EPSP, the ganglion was perfused with15mMMg2+ saline to eliminate chemical synaptic transmission(Zipser, 1979) for 15 min before the first test of the electricalsynaptic transmission. Following an initial test of the electricalEPSP, the ganglionwas perfused for 15minwith CNQX or AMPA(Sigma; St. Louis, MO) dissolved in 15 mM Mg2+, followed by a15 min washout in 15 mM Mg2+ saline. In some experiments,CNQX/AMPA treatments were made in the presence of con-canavalin A (Con A; Sigma), dynamin inhibitory peptide (DIP) orpep2-SVKI (Tocris; Ellisville,MO). In the case of DIP or pep2-SVKI,the peptides were dissolved in the electrode filling solution andinjected into the cell via ionotophoresis (1 nA of negativecurrent) for 10 min prior to the start of the experiment.
To examine theeffect of CNQXonelectrical coupling betweenpairedRetzius cells (R cells) or paired longitudinalmotor neurons(L cells), 500 ms current pulses (−0.25, −0.5, −0.75, −1, −2, −3 nA,0.25, 0.5, 0.75, 1.0 nA,) were injected into one Retzius cell (R1) or Lcell (L1) and the resulting changes in membrane potential in R1/L1cell (V1) and thecontralateralR2/L2cell (V2)were recordedandmeasured. Electrical coupling between these pairedneuronswascalculated as the ratio V2/V1. Electrical coupling was measuredprior to and following a 15 min treatment with 20 μM CNQXdissolved in leech normal saline solution.
To determine whether synaptic activity could producemodulation of the chemical and electrical components of theT-to-T synapse, low frequency stimulation (LFS) was used toinduce long-term depression (LTD) at T-to-T synapse. LFS-inducedLTDwas carried out in normal saline and in 0mMMg2+
saline which allowed for separation between electrical andchemical EPSP (see Results; Baylor and Nicholls, 1969). Follow-ing an initial pre-LFS recording of the EPSP, the T-to-T synapseunderwent LFS training by stimulating the presynaptic T cell toelicit a single action potential 900 times at 1 Hz, a commonprocedure for inducing LTD (Anwyl, 2006). The T-to-T EPSPwasre-tested 40 min after LFS.
EPSP amplitude and input resistance measurements werenormalized to their initial values (% of baseline) and presentedas the mean ± SE. In experiments conducted in saline with15 mM Mg2+ the baseline measurement for EPSP amplitudeand input resistance were taken after the ganglion had beenbathed in the high Mg2+ saline for 15 min. Statistical analyseswere performed using two-way ANOVA and Newman–Keulspost hoc tests (Statistica analysis software; Statsoft).
Acknowledgments
The authors thank Drs. Brenda Moss and Kevin Crisp for theirhelpful comments while this manuscript was being prepared.
Supported by grants from the National Science Foundation(IBN-0432683, BDB), the Advisory Council for the South DakotaSpinal Cord/Traumatic Brain Injury Research and by asubproject of the National Institutes of Health grant (P20RR015567, BDB), which is designated as a Center of BiomedicalResearch Excellence (COBRE).
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