gap junctions as electrical synapses

Journal of Neurocytology 26, 349–366 (1997)

Gap junctions as electrical synapsesM I C H A E L V. L . B E N N E T T

Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY10461, USA

Received 26 November 1996Supplement to 25th Anniversary Issue

Summary

Gap junctions are the morphological substrate of one class of electrical synapse. The history of the debate on electrical vs.chemical transmission is instructive. One lesson is that Occam’s razor sometimes cuts too deep; the nervous system does itsoperations in a number of different ways and a unitarian approach can lead one astray. Electrical synapses can do many thingsthat chemical synapses can do, and do them just as slowly. More intriguing are the modulatory actions that chemical synapsescan have on electrical synapses. Voltage dependence provides an important window on structure function relations of theconnexins, even where the dependence may have no physiological role. The new molecular approaches will greatly advanceour knowledge of where gap junctions occur and permit experimental manipulation with high specificity.

Introduction

A synapse can be defined as a specialized site offunctional interaction between neurons. By this defini-tion gap junctions form one class of electrical synapse(Bennett, 1977). Another kind of electrical synapsemediates short latency inhibition of the Mauthner cellof teleost fishes and possibly mammalian cerebellarPurkinje cells; this form of electrical transmission isnot mediated by gap junctions, and involves differentjunctional specializations (Faber & Korn, 1989). In ad-dition, there probably are electrical interactions thatoccur between closely apposed cells without obviousgap junctions or specialization other than the absenceof interposed glia (Faber & Korn, 1989; Jefferys, 1995).Whether these sites are to be considered synapses orincidental or accidental sites of interaction may be-come clear with greater knowledge of the develop-mental mechanism.

One reason for asserting at the outset that gapjunctions between neurons are synapses is to avoidsome of the contradictory statements in the literature.For example, J. G. R. Jefferys (1995) in his recentarticle in the Physiological Review considers ‘‘fourclasses of nonsynaptic interaction, mainly in themammalian brain’’ of which the first is ‘‘Electrotonic(and chemical) coupling through gap junctions’’. Yethe also writes of ‘‘ ‘gap junctions,’ which commonlyserve as electrical synapses in invertebrates but appearto be used less often for electrical signalling in verte-brates’’. The relative incidence of chemically transmittingand gap junction synapses in mammalian brains, aswell as brains of earlier diverging vertebrates andinvertebrates is not clearly resolved, as discussed below.

0300–4864/97 ( 1997 Chapman and Hall

Although most of the data summarized here ante-date the cloning of connexins, the discussion will besimpler if some of the newer basic facts are accepted(Bennett et al., 1991, 1994; White et al., 1995a). Gapjunctions in vertebrates are made of proteins termedconnexins that are encoded by a gene family. A junc-tional channel consists of two hemichannels (or con-nexons) in series, one provided by each of the apposedcells. Hemichannels, which are hexamers of connexin,can be homomeric, i.e., comprised of a single type ofconnexin. It is likely that some hemichannels are het-eromeric (Stauffer, 1995). Junctions can be homotypic,i.e., formed by two hemichannels of the same kind,and they can be heterotypic, i.e., formed by hemichan-nels of different kinds or in earlier usage formed bydifferent cell types, which we now know may expressthe same or different connexins. A given cell type canexpress more than one connexin. A given connexin canbe expressed by more than one cell type. Connexinsexpressed by neurons and forming electrical synapsescan also be expressed by other cells. Thus, gap junc-tions between inexcitable cells can serve as models forelectrical synapses.

Some history

A prominent controversy in early Neuroscience waswhether synaptic transmission was chemical or electri-cal, a dichotomy sometimes affectionately character-ized as ‘soup or sparks’. After a long period of debatebetween pharmacologists and electrophysiologists,each group having not quite convincing arguments

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(applied chemicals mimic the effect of nerve stimulation,nerve stimulation releases epinephrine or acetylcho-line; chemicals would be too slow to mediate transmis-sion in the CNS; the action potential is propagatedelectrically and should be able to cross the synapse inthe same way, so who needs chemicals), it becameclear that CNS inhibition and neuromuscular trans-mission were chemically mediated. The pendulumswung, dragging with it the generalization to excita-tory transmission between neurons, although here theinitial data were much less convincing. Nevertheless,Paul Fatt, and no doubt Bernard Katz, were aware thatsynapses between large fibre systems might well beelectrical (on the unreliable assumption that large sizemeans large conductance, see Fatt, 1954), and in a pa-per that almost everyone knows about (but few haveread) Edward Furshpan and David Potter reportedfrom Katz’s laboratory that fast excitatory transmis-sion in the crayfish giant fibre system was electricallymediated, although there were also chemically me-diated inhibitory inputs (Furshpan & Potter, 1959).Independently and more or less simultaneously on theother side of the world Akira Watanabe reported, ina paper that almost no one knows about, that neuronsin the cardiac ganglion of the mantid shrimp, Squilla,were electrically coupled (Watanabe, 1958). He sug-gested that the coupling was responsible for syn-chronous firing (certainly right) and was mediated bycytoplasmic continuity of axonal processes (probablywrong). In the crayfish there is fast transmission forescape responses and rectification in the junctionalmembrane, so that impulses cross the giant to motorsynapses in only one direction. In the shrimp there isslow, potentially bidirectional transmission leading tosynchronization of motoneuron activity and cardiaccontraction. Thus, electrical transmission is mediatingtwo quite different functions: (1) transmitting excita-tion from an active axon to a postsynaptic cell, whichcan be quite similar at electrical and chemical synapses(Fig. 1A), and (2) synchronizing activity of cell bodies,in which coupling is both excitatory to the less de-polarized cell and inhibitory to the more depolarizedcell, since current flowing to depolarize one cell ismaking the other cell less depolarized (Fig. 1B, C).Synchronization might also be mediated by reciprocalchemical mediated excitation between cells.

At this stage one of the arguments for chemicaltransmission was the large size of the postsynapticpotential at the neuromuscular junction and the smallsize of the presynaptic terminal. Chemical transmis-sion appeared to permit amplification and on the faceof it an electrical synapse with passive junctionalmembrane did not. This argument is invalid, as can beseen by consideration of the molecular anatomy.The postsynaptic potential (PSP) is generated by theacetylcholine receptors (AChR) densely packed in thesubsynaptic membrane. If one were to substitute

Fig. 1. Electrical and chemical synapses, diagramatically.(A) Axosynaptic synapses. Cell 1 forms a chemical synapseon cell 3. Arrows indicate current flow if the synapse isexcitatory (e) or inhibitory (i). Cell 2 forms an electricalsynapse on cell 3. During the presynaptic impulse Na] flowsinto the terminal and K], the main charge carrier in thecytoplasm, flows through gap junction(s) into the postsynap-tic cell. The initial segment, where the impulse arises, hasdifficulty distinguishing the mode of excitatory transmis-sion. (B) Dendrodendritic synapse mediating electrotoniccoupling and synchronous activity of neuronal somata. If thearrow indicates the direction of current flow, the cell at thearrow head is excited and the cell at the tail is inhibited; thus,the synapses are both excitatory and inhibitory. (C) Coup-ling of cell bodies by way of presynaptic fibres can serve thesame synchronizing function as dendrodendritic junctions.

gap junction channels connecting to the presynapticterminal one to one for the AChR molecules and puta high density of Na channels in the presynaptic ter-minal, one could also generate a large PSP (Fig. 1A).The action potential mechanism is itself an amplifierand amplification could be provided by the terminalaxon as well as by the subsynaptic membrane. In anycase, the size argument would apply to only a fewsynapses where the PSPs are very large.

At essentially the same time, Stanley Crain, HarryGrundfest and I were working on the pufferfish,a teleost with large neurons that sit on top of the spinalcord just behind the cerebellum (Fig. 2) (Bennett et al.,1959a). Back before patch clamping, large accessibleneurons were particularly attractive. These so calledsupramedullary neurons are monopolar and have ex-tensively branching axons that run out through thedorsal roots to the skin. They are effector cells, buttheir action is still unknown. What is relevant to thisreview is (1) they are electrically coupled via gapjunctions (Bennett et al., 1967a) (the ultrastructure wasdone by Yasuko Nakajima and George Pappas), and(2) they fire synchronously (Bennett et al., 1959b), but(3) the degree of synchronization is not very precise,a point to which I will return (Fig. 3). It is a somewhatself serving aside to point out recent evidence that gap

Gap junctions as electrical synapses 351

Fig. 2. Anterior spinal cord of the puffer viewed from the dorsal side. The posterior limit of the cerebellum is to the left. Thesupramedullary neurons are the large round cells, about 250 lm in diameter, that are located on the surface of the cord.Several of the cells that had been penetrated for intracellular recording are dark because of increased Toluidine Blue staining.From Bennett et al., 1959a.

junctions in the nematode, mollusc, arthropod line andthe vertebrate line are made of unrelated proteins,although there are numerous convergent features (seeBarnes, 1994). Thus, we were the first to identifyconnexin based, vertebrate electrical synapses.

Furshpan and Furukawa were not far behind. Inextending the ideas that escape systems need shortlatency and perhaps that large size suggests electricaltransmission, they found electrical transmission at theclub endings on the goldfish Mauthner cells (Furshpan& Furukawa, 1962). These cells mediate an escape tailflip, and the synapses on them were well known at thelight microscope level from the work of David Bodian(1938). They also described the electrical inhibitorytransmission that tends to keep the Mauthner cellsfrom firing simultaneously (Furukawa & Furshpan,1993).

Electrical coupling in neural control of electric organs

In collaboration with Harry Grundfest a number of uswere working on mechanisms by which the electricorgans of fishes generate electricity (Bennett, 1971),and I and several others went on to look at neuralcontrol of the discharges (Bennett, 1968). The problemwas particularly interesting, because in most of these

fishes the discharges are brief and in some the fre-quency of discharge is very high, greater than 1 kHz.Both properties require highly synchronous activationfor reasonable efficiency. As an example, consider theelectric catfish. Its electric organ, which is found in theskin, is controlled by only two neurons, one on eitherside of the medulla (Bennett et al., 1967b). Gradedstimulation of afferents produces smoothly gradedEPSPs in them, but always excites them together; nostimulus can be found to excite one at a time (Fig. 4A).The reason is that if one cell fires, its action potentialpropagates through electrical synapses to excite theother (Fig. 4B; the spread of hyperpolarization be-tween cells is shown in Fig. 4C). Here and in manyother electric fishes, electrical transmission betweenneurons is required because of its speed; the electricorgan discharges are so brief, in this case &1 ms, thatchemical transmission with a 0.5 ms delay betweenneurons would not give adequate precision of firing.Reciprocal excitation is necessary for all the control-ling cells to fire together; to get really precise syn-chronization the excitation must be electrical. In thissituation the speed and reciprocity of electricalsynapses are both important. Several other points areillustrated here, given the additional evidence that

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Fig. 3. Synchronous activity in four supramedullary neurons diagrammed in D. The most rostral cell is shown on theuppermost trace. Electrical stimuli were given to the cranial nerves (A

1–A

3) and cauda equina (B

1–B

3); the skin was stimulated

tactilely (C). Weak, near threshold stimuli in A1

and B1; strong stimuli at the same sweep speed in A

2and B

2and at a slower

sweep speed in A3

and B3. Calibration pulses (50 mV, 1 ms) occur at the beginning of each of the four traces followed after

1 ms by the electrical stimulus artifact. All cells generate the same number of impulses, although the action potential fails toinvade the somata in some cases. Impulses tend to arise earlier rostrally for the cranial nerve stimulus and earlier caudally forthe cauda equina stimulus. From Bennett et al., 1959b.

transmission to the electromotor neurons is via gapjunctions and that the cells are coupled to each othernot directly but rather by way of presynaptic fibresthat end on both of them (Fig. 1C, other electromotorneurons are coupled via dendrodendritic gap junc-tions as in Fig. 1B). An impulse in one or a few afferentfibres will not excite either cell. As stimulus strength isincreased, both cells are depolarized; when one ofthem reaches threshold, the other cell is already nearthreshold and the impulse rapidly propagates be-tween them (Fig. 4A). An action potential evoked bydirectly stimulating one cell takes a longer time toexcite the other cell (Fig. 4B). Thus, weak couplingbetween a single afferent and the postsynaptic cell canbe very effective when enough afferent fibres are ac-tive. Also, these electrical synapses are unidirectionalwith respect to impulse propagation, since impulses inone or a few afferents will not excite the electromotorneurons, but an impulse in one electromotor neuronwill excite most or all of the afferents. This unidirec-tional action arises because the input resistances look-ing away from the synapses on the two sides aredifferent; the junctions themselves are electrically

symmetrical and essentially linear. Actually, mostsynapses, acting alone, do not mediate impulse propa-gation in either direction, so the classic synaptic prop-erty of unidirectional action can readily occur atelectrical as well as chemical synapses, even neglect-ing the possibility of rectification in the junctionalmembrane.

From a teleological perspective, one might arguethat a single cell command nucleus would not requireelectrical synapses; that appears to be a valid argu-ment, and the electric catfish command nucleus isdown to only two cells. However, electrical synapsesallow multiple cells to act with nearly the precision ofa single cell, and having multiple cells in parallelreduces the number of efferent synapses that a singlecell must support.

Once a central ‘command nucleus’ has initiated asynchronous signal for the electric organ to discharge,other adaptations are involved in achieving simulta-neous activation of different regions of the electricorgan, which may be located at quite different distan-ces from the command nucleus (Bennett, 1968). Theremay be ‘relay nuclei’ intercalated between command


Fig. 4. Properties of the giant electromotor neurons of the electric catfish, Malapterurus electricus. (A) Upper and lower traces,recordings from right and left cells, respectively. Brief stimuli of gradually increasing strength are applied to the nearbymedulla (several superimposed sweeps; the stimulus artifact occurs near the beginning of the sweep). Depolarizations ofsuccessively increasing amplitude are evoked until in one sweep both cells generate spikes. (B) Two electrodes in the right cell,one for passing current (shown on the upper trace) and one for recording; one recording electrode in the left cell. The lowertraces, which are from the recording electrodes, start from the same base line. When an impulse is evoked in the right cell bya depolarizing pulse, the left cell also generates a spike after a short delay. (C) When a hyperpolarizing current is passed in theright cell, the left cell also becomes hyperpolarized, but more slowly and to a lesser degree (display as in B). (D) When organdischarge is evoked by irritating the skin, a depolarization gradually rises up to the threshold of the giant cell and initiatesa burst of three spikes (lower traces, base line indicated by superimposed sweeps). Each spike produces a response in theorgan (upper trace, recorded at high gain and greatly reduced in amplitude because curare, used to prevent movement, alsoblocked transmission from nerve to electrocyte). Thus, these two cells comprise the command nucleus. From Bennett et al.,1967b.

nucleus and electromotor neurons, which innervate(or in one group comprise) the generating cells; therelay and electromotor neurons commonly are electri-cally coupled, which would tend to synchronize theirfiring and correct for any loss of synchronization thatarose in conduction of the signal from the commandnucleus. The general solution to the problem of differ-ing distances is to slow down conduction in the path-way to the more proximal regions of the organ bymaking the fibres going to that region have a smallerdiameter (and perhaps an internodal distance shortenough to reduce conduction velocity; Meszler et al.,1974) and/or by making the fibres take a more deviousroute. In the electric eel, for example, the anterior andposterior extremes of the organ may be separated byover 1 m and at least 10 ms conduction time. Spinalelectromotor neurons, which innervate the electro-cytes, are activated by spinomedullary fibres from arelay nucleus in the medulla (Meszler et al., 1974).Transmission from the descending fibres is electrical,rise times of the EPSPs are short, and the compensatory

delays are primarily if not exclusively in conductiontimes in the preterminal fibres and the peripheralnerves to the electric organ. Similar mechanisms mayoperate in the auditory system where very precisearrival time comparisons are made to permit sourcelocalization (Carr & Boudreau, 1993).

Electrical coupling in motor systems

With the insight that a fast synchronous responserequires electrical transmission, we extended ourstudies to motor activity, starting with the sonic motorsystem of the toadfish, Opsanus tau. As in a number ofindependently evolved species, this fish generatessound by rapid synchronous contractions of its swimbladder musculature. Obviously, out of phase musclefibres reduce the amplitude of the contraction. In thebreeding season, males generate ‘boat whistle’ calls inwhich the fundamental frequency generated by repeti-tive contractions of the swim bladder muscle is about200 Hz. Obviously, muscle fibres contracting out ofphase reduce the amplitude of oscillation. The sonic

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motoneurons proved to be electrically coupled by gapjunctions, both by dendrodendritic junctions and byway of presynaptic fibres (Pappas & Bennett, 1966).

Control of the pectoral fins of the hatchetfish,Gasteropelecus, was another example. This fish uses itspowerful pectoral fin adductor muscles to jump outof the water in an escape response mediated by theMauthner cell (Fig. 5, diagram). They were also re-ported to flap their fins repetitively to taxi along thewater surface. The pectoral fin motoneurons are in-deed electrically coupled. Also, they are excited elec-trically by large interneurons termed giant fibres. Thegiant fibre to motoneuron synapse is electrical, whichshould reduce the latency of the pectoral fin response(Fig. 5A), and it is rectifying, which permits activationof subsets of motoneurons by independent inputswithout propagation into the giant fibres and excita-tion of the entire system (Auerbach & Bennett, 1969b;Hall et al., 1985). However, the Mauthner cell to giantfibre synapses are chemically transmitting and uni-directional, where a rectifying synapse might havesaved 0.3 ms in response time (Fig. 5B) (Auerbach &Bennett, 1969a).

Extension to mammals of the concept that syn-chronous or short latency responses should utilizeelectrical coupling was not very successful. Henri

Korn and colleagues did find electrical inputs to vesti-bular neurons in the rat, perhaps functionally similarto the club endings on the Mauthner cell (Korn et al.,1973), but apparently these synapses are not electricalin larger mammals, such as cat. One possible reason isthat in a large animal nerve conduction distances andinertia of the body parts are so great that a responsecannot be rapid enough to require electrical transmis-sion, particularly since chemical transmission is fasterin warm blooded animals. Another site of gap junctionmediated electrical transmission in mammals is themesencephalic nucleus of the fifth cranial nerve (Baker& Llinas, 1971), where the electrical mode of transmis-sion was first predicted from morphological observa-tions of gap junctions (Hinrichsen & Larramendi,1968). In rodents mastication is fast and synchronous,but it would be difficult to conclude that electricaltransmission were required for its speed. Although itis true in principal that ‘‘Direct electrical interactionscan alter neuronal activity on a shorter time scale thanchemical synaptic transmission’’ it is not true that‘‘there is much (italics mine) less synaptic delay asso-ciated with electrical interactions’’ (Valiante et al.,1995), and the delay at a gap junction synapse can beconsiderably longer than the minimum at a chemicalsynapse because of the time to charge the postsynaptic

Fig. 5. Minimum synaptic delays at electrical and chemical synapses in the hatchet fish, Gasteropelecus. (A) Electrical EPSP atthe rectifying electrical synapse between a giant fibre and a pectoral fin motoneuron. (B) Chemical EPSP at the synapsebetween a Mauthner and a giant fibre evokes an impulse in the giant fibre. Arrows indicate onset of the Mauthner cell impulseand the EPSP. The lower trace in each record shows the stimulating current applied in the presynaptic element. The diagramshows lateral and frontal views of the fish. The spinal cord, brain, pectoral fin adductor muscle and its innervation areindicated. mf: Mauthner fibre, gf: giant fibre, mn: motoneuron. From Auerbach & Bennett, 1969a, b.


membrane capacity (Bennett, 1966). In addition, therecan be delays in presynaptic fibres due to conductionof the impulse, which is electrical. As noted above,conduction delays provide most if not all of the com-pensatory delays for electric organ synchronization(and may be involved in auditory localization).

Firing of the supramedullary neurons of the puffer-fish is not highly synchronous (Fig. 2), and reciprocalexcitation mediated by chemical synapses wouldprobably be able to provide an adequate degree ofsynchronization; gap junctions may do it better be-cause subthreshold potentials are transmitted, and allneighbouring cells tend to reach threshold at the sametime. If one really pushes functional arguments, bothchemical and electrical transmission could be made towork at most synapses (Bennett, 1977). Where electri-cal synapses are clearly better is in speed and in reci-procity (Fig. 1). Where chemical synapses are clearlybetter is in inhibition and possibly temporal changesas a result of prior activity (see Pereda & Faber, 1996).Furthermore, chemical synapses can have G-proteincoupled or metabotropic receptors. To be sure, gapjunctions can transmit small molecules in addition toions and in this manner mediate chemical communica-tion between cells (Bennett et al., 1991). A chemicalsignal might be generated in a presynaptic terminalby, e.g., Ca2] entry through a voltage dependent chan-nel, but the many G protein initiated cascades weigh infavour of chemical transmission. The early hypothesisthat gap junctions mediate developmental gradientshas not yet been borne out. It appears that most sig-nalling molecules are peptides that act extracellularly.Gap junctions are implicated in the transmission ofCa2] waves that propagate between glia and mayinvolve neurons (Dani & Smith, 1995; Sanderson,1995), but Ca2] waves can also be propagated byextracellular signals in what is clearly chemical trans-mission (Osipchuk & Cahalan, 1992; Hassinger et al.,1996).

How many electrical synapses are there?

By the early 1960s, it was clear that there were elec-trical as well as chemical synapses. Eccles (1964) didsuggest, wrongly, that electrical synapses would notbe found in mammals. But it is difficult to determinehow many electrical synapses of the gap junction typethere are. Although a very short synaptic delay indi-cates electrical transmission, as noted above, the delayat electric synapses can exceed the delay at chemicalsynapses, particularly at mammalian body temper-ature (Bennett, 1977). Direct measurement of electro-tonic coupling by simultaneous recording from pairsof neurons remains difficult, except where cells arelarge. Patch clamping has made it easier, but it is stilldifficult to record from presynaptic fibres. Dye coup-ling can be convincing, but a negative result is not, and

some positive results may be artifactual. Conversely,reversal by polarization is diagnostic of a chemicallymediated PSP, and usually is easily done for inhibi-tion. It may be hard to reverse a chemically mediatedEPSP, particularly if the synapses are located on thedendrites distant from the recording site. Now thatglutamate is identified as the most common CNS ex-citatory transmitter, pharmacological approaches aremore feasible, but failure of glutamate antagonists toblock transmission would not be considered verystrong evidence for electrical transmission. Low Ca2]

high Mg2] solutions block chemical transmission, butdo not distinguish between mediation by gap junc-tions and field effects (Dudek et al., 1986).

Anatomical methods including electron micro-scopy, immunostaining and in situ hybridization allhave their problems. Electron microscopy requiresgood fixation, not always easy in CNS tissues, and gapjunctions that are small compared to section thickness,perhaps five junctional particles across, are difficult toidentify. Freeze fracture permits identification of smalljunctions, but in neuropil it may be difficult to deter-mine the identity of the structures forming them. A lab-orious combination of confocal light microscopy andfreeze fracture electron microscopy has revealed anunexpectedly high incidence of synapses with bothgap junctions and morphological characteristics ofchemical synapses in rat spinal cord (Rash et al., 1996).Presynaptic vesicles, which define an active zone, arealso found at axosomatic and axodendritic synapseswhere electrophysiological findings indicate thattransmission is purely electrical (Pappas & Bennett,1966; Bennett et al., 1967b; Korn et al., 1973). In fact, itappears that all axodendritic and axosomatic synapseswith gap junctions also have active zones, i.e., they aremorphologically mixed synapses. Dual electrical andchemical transmission is not that common (Martin& Pilar, 1963; Christensen, 1983; Lin & Faber, 1988).The presynaptic vesicles and densities at functionallyelectrical synapses may be involved in membrane re-cycling of surface proteins or uptake of extracellularfactors. Conversely, correlation of the number of gapjunction channels at club endings on the Mauthner cellwith junctional conductance suggests that most of thechannels are closed and that gap junctional area is nota good measure of junctional conductance (Tuttle et al.,1986). Also, single channel conductance of junctionsformed by cloned connexins can vary by an order ofmagnitude (Veenstra et al., 1995). Antibodies to manyconnexin proteins are not yet available, and presenceof mRNA shown by in situ hybridization does notguarantee protein synthesis, nor does protein syn-thesis guarantee junction formation. My summaryconclusion based on currently available data is thatgap junctions are a small minority of synapses in thebrain but that there are probably a few more still tobe found. I believe this characterization applies to

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mammals and earlier diverging vertebrates and also toanimals of the arthropod line. (Here I use gap junctionfor both mammal and arthropod lines in the same wayI would use wing for the analogous structures on birdsand insects.)

The widely held opinion that electrical transmissionis characteristic of lower forms probably derives fromthe large cell systems that were studied in the initialperiod of intracellular recording, which hardly consti-tute a reasonable sample. There may be a kernel oftruth in the idea, since synaptic delay is shorter atmammalian body temperature and the advantage ofelectrical transmission is less. In many of the sitesof gap junction mediated electrical transmission inmammalian CNS, such as olfactory bulb (Pinching& Powell, 1971), sensory motor cortex (Sloper, 1972;Peinado et al., 1993), hypothalamus (Hatton & Yang,1994), inferior olive (Llinas et al., 1974), cerebellum(Sotelo & Llinas, 1972) and retina (Vaney, 1991), thefunctional advantages are more likely to be in reci-procity and transmission of subthreshold potentials.With respect to primitiveness, one can argue that uni-cellular organisms evolved the basic machinery of

chemical transmission for release of and response tochemicals, but have no functional equivalent of elec-trical synapses. Thus, gap junctions are the more ad-vanced form of transmission. As more genomes oflower life forms are sequenced, it may become clearerhow gap junction forming proteins evolved. Many ofthe proteins recently implicated in neurotransmitterrelease have homologues involved in secretion inyeast (Calakos & Scheller, 1996), but homologues ofconnexins have not been reported outside of vertebrates(Mushegian & Koonin, 1993).

Flexibility in electrotonically-coupled systems

Although in a number of reviews I emphasized thepossible similarities in function of chemical and elec-trical synapses (e.g., Bennett, 1977, 1985, Fig. 1A), mymain point was that one needed to examine the modeof transmission carefully at new sites rather than as-sume that it was chemical. There was also self interestin that I did not want to be working on a second classor more primitive synapse. The real stretch in arguingfor basic equality of electrical and chemical transmission

Fig. 6. Summation and facilitation of electrical EPSPs evoked by antidromic stimulation of the electrically coupled elec-tromotor neurons of the stargazer, Astroscopus. Upper trace: monopolar recording of the antidromic volley. Lower trace:intracellular recording from four different electromotor neurons. (A–C) Superimposed sweeps of pairs of stimuli given withdifferent delays of the second stimulus. The second response summates with the first response and is increased in amplitude.Faster sweep in B and C. (D) A tetanus shows extensive summation and facilitation. Superimposition of two sweeps with anadditional stimulus in one makes clearer the maximum degree of facilitation near the end of the tetanus. From Bennett& Pappas, 1983.


was with respect to synaptic plasticity. Gap junctionsexhibited very little modifiability or dependence onprior activity, in part perhaps because they were beingstudied in systems where synchrony and constancywere important. One could demonstrate temporalsummation of electrical PSPs, which depends oncharging the postsynaptic membrane capacity, andthere were several examples of temporal changes thatwere unexpectedly long lasting. For example, in ourinitial studies of the puffer, stimulation of one supra-medullary neuron would usually not excite the others;however, paired stimulation sometimes would; theeffect of the first stimulus could last for several hun-dred milliseconds, a duration that we thought in-dicated chemical mediation (Bennett et al., 1959b). Welater showed that the facilitation was due to a longlasting depolarizing afterpotential that facilitatedpropagation across the electrical synapses (Bennett,1966). In the stargazer, another electric fish, antidromicstimulation evokes electrical PSPs in the electromotorneurons (which are derived from the oculomotor

nucleus). These PSPs show pronounced summation andfacilitation with repetitive stimulation; the facilitationis due to more extensive invasion of the antidromicimpulses closer to the sites of electrical coupling inthe dendrites (Fig. 6) (Bennett & Pappas, 1983). Theseexamples indicated that electrical synapses can exhibitactivity dependent changes in effectiveness of unex-pectedly long duration, although there was no indica-tion that the phenomena were functional at these sitesor at all common in nervous systems in general.

We then found that transmission at electricalsynapses can be under the control of chemicalsynapses on the same cells. We first observed thisphenomenon in the mollusc, Navanax. This animal canexpand its pharynx very rapidly as an ingestive re-sponse that sucks in prey. Under resting conditions,the pharyngeal motoneurons innervating the expan-sion musculature are electrically coupled (Fig. 7A, B)and their firing tends to be synchronous, which ac-counts for the rapid pharyngeal expansion (Spira et al.,1980). However, stimulation of the pharyngeal nerve

Fig. 7. Synaptic control of electrical coupling of expansion motoneurons in the opisthobranch mollusc, Navanax inermis. (A, B)Upper trace: recording from a medium sized expansion motoneuron (M) in the buccal ganglion on one side. Middle trace:recording from the giant expansion motoneuron (G) in the same ganglion. Lower trace: current applied in one or the other cell.(A) Hyperpolarization due to current applied in the giant cell spreads to the medium sized cell. (B) Hyperpolarization due tocurrent applied in the medium sized cell spreads to the giant cell (higher gain in the giant cell recording). The couplingcoefficient for spread from M to G cell is less than that for spread from M to G cell, because the G cell is of lower resistance.(A@, B@) When the pharyngeal nerve is stimulated at the beginning of the sweep, which produces a &10 mV depolarization forabout 150 ms and then a smaller long lasting depolarization, electrical spread between the two cells is blocked. During theperiod of block, the input resistance of the cells is reduced. Diagram: inhibitory inputs (filled circles) along the couplingpathway short circuit electrical spread between the cell bodies; these inputs reduce synchronization and excitability but do notprevent independent firing of the cells by excitatory synapses (Ys) located near the somata. From Spira & Bennett, 1972.

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or stimulation of the pharyngeal wall reduces thecoupling between them (Fig. 7A@ & B@) (Spira & Be-nnett, 1972). The loss of coupling appears to be due toinhibitory synapses along the pathway connecting theneurons; increase in conductance simply short circuitsthe electrotonic spread. Under these circumstances,the neurons can be activated asynchronously by otherexcitatory inputs that synapse on the motoneuronscloser to the site of impulse initiation. Our functionalexplanation of these data was that all the expansionmotoneurons should be active for prey ingestion butthat during peristaltic swallowing expansion shouldinvolve subsets of the expansion musculature actingsequentially. Similar inhibitory control of coupling hasbeen suggested for the mammalian inferior olive(Llinas et al., 1971). In this nucleus dendrodendriticgap junctions synchronize firing, but inhibitory inputslocalized to the dendrites are in a strategic position toshortcircuit the coupling and permit asynchronousfiring to somatic inputs.

A further variant was provided by goldfish ocu-lomotor neurons; these cells are coupled in the cell

body region, and synapses depolarize this region tomediate the relatively synchronous actions of eyewithdrawal and perhaps saccadic eye movements(Fig. 8B1 & B2) (Kriebel et al., 1969; Korn & Bennett,1975). Vestibular inputs that mediate the slow phase ofnystagmus evoke responses that arise abruptly fromthe baseline (recorded in the soma); these inputs arelocated out on the dendrites where the cells presum-ably are not coupled (Fig. 8A1 & A2, inset). In this caselocalization of inputs to regions that are and are notcoupled appears to determine the degree of functionalcoupling and whether firing is synchronous or asyn-chronous. Weak coupling of the somata would permitasynchronous firing of impulses arising in the den-drites, but this same coupling would synchronizefiring if the somata were depolarized by other inputs orif many of the cells were active at about the same time.

A dramatic and distinctly different form of synapticcontrol of coupling was observed in the turtle andteleost retina (Teranishi et al., 1983; Piccolino et al.,1984). In the unstimulated preparation, dye couplingbetween horizontal cells is extensive, and dye injected

Fig. 8. Dendritic and somatic impulse initiation sites in teleost oculomotor neurons permit different degrees of coupling.Upper trace: intracellular recording from a medial rectus motoneuron of the puffer, Spheroides maculatus. Middle trace: appliedcurrent applied in the cell. Lower trace: efferent activity in the medial rectus nerve. (A

1, A

2) Response to stimulation of the

ipsilateral VIIIth nerve at a fixed stimulus strength. (A1) The impulses arise from a nearly level baseline. (A

2) Hyperpolariz-

ation delays the first response and reduces the number of impulses, but there is little PSP at the times that the first two spikesarose in A

1. These data indicate that the impulses are generated at some distance from the recording site. (B

1, B

2) Stimulation

of the ipsilateral ophthalmic nerve at a fixed strength that would produce eye withdrawal. (B1) The first impulse arises from

a slowly rising EPSP (arrow); later impulses arise from successively lower levels of depolarization. (B2) Hyperpolarization

blocks the evoked impulses leaving a large slowly rising EPSP (two superimposed sweeps with and without nervestimulation). The diagram on the right gives the suggested localization of synaptic inputs underlying these responses.Dendritic inputs (left arrow) are activated by stimulation of the ipsilateral eighth nerve. There is no coupling, and movementsare smoothly graded in amplitude; impulses in the somata arise abruptly from the baseline. Somatic inputs (right arrow) areactivated by stimulation of the ophthalmic nerve or contralateral eighth nerve. There is weak coupling between the cell bodiesby way of the presynaptic fibres, which results in some increase in synchronization; impulses in the somata arise from obviousEPSPs. From Kriebel et al., 1969.


into one cell spreads to many neighbours. Appli-cation of dopamine to the retina greatly restricts dyeand electrical coupling. Subsequently, dopamine andcAMP increasing agents were shown to decrease junc-tional conductance and dopaminergic interplexiformcells are thought to be the endogenous source (Dowl-ing, 1991). These cells are presumed to be active dur-ing light adaptation, when they mediate the observedreduction in receptive field size. This pronouncedmodulation of junctional conductance may be due tophosphorylation of the channel protein, itself. Theconnexins in the retinas of these animals have not yetbeen identified. Rodent Cx32 has a phosphorylationsite for cAMP dependent kinase, but the effect ofphosphorylation is to increase rather than decreasecoupling (Saez et al., 1990). Similar phenomena mayoccur in the mammalian retina (Hampson et al., 1994),which has many electrical synapses (Vaney, 1991).

Extensive coupling between cortical neurons in theneonatal rat has been observed using a gap junc-tional permeable tracer, neurocytin (Fig. 9) (Peinadoet al., 1993). This communication largely disappearsduring development, and it is hypothesized to be in-volved in synapse formation. In the young animalapplication of dopamine to brain slices reduces thetracer coupling (Rorig et al., 1995), but the relation, if

any, between this process and the loss of couplingwith maturation is unclear.

More recently Faber and colleagues (Silva et al.,1995; Pereda & Faber, 1996) showed LTP of electricalPSPs at club endings on the Mauthner cell. The phar-macology suggests involvement of NMDA receptorsas in some forms of mammalian LTP. Increase injunctional conductance may involve hemichannels ineither pre- or postsynaptic elements, since most of thegap junction channels appear to be closed under nor-mal conditions (Tuttle et al., 1986) and may be closedby either the hemichannel in the Mauthner cell orafferent fibre or by both.

The incidence of gap junctions certainly has all thecontrols of other cell proteins. In different experi-mental systems controls have been demonstrated attranscriptional, translational and posttranslationallevels (Bennett et al., 1991). Posttranslational modifica-tions include phosphorylation and myristylation, andthe mechanisms of connexin assembly, membrane in-sertion and junction formation are subjects of activeinquiry. In some culture systems at least, turn over israpid and removal of junctions is a possible if slowform of modulation of junctional conductance. Coup-ling between neurons may change as a function ofhormonal state (Hatton & Yang, 1994).

Fig. 9. Coupling of cortical neurons varies with age. Neurobiotin injected into a single cell in neocortical slices spreads tovarying numbers of neighbours. (A) At postnatal day 5 many clusters of labelled neurons consist of vertically orientedcolumns spanning several layers. Approximate layer boundaries are shown on the right. Dashed lines indicate the region inwhich there are labelled neurons. (B) At postnatal day 7 coupling is restricted to cells in the immediate vicinity of the injectedcell’s dendritic tree. (C) By postnatal day 15 many fewer cells are labelled, suggesting a decreased level of gap junctionalcoupling. Cells in B and C are in layer 2/3. Scale bar\50 lm. From Peinado and colleagues, 1993.

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Ca2], H] and extrinsic modulators of junctionalconductance

Experimental modulation of junctional conductancewas an early goal. Treatments that increased intracel-lular Ca2] decreased junctional conductance in insectsalivary gland cells, but Ca2] levels were not accu-rately measured (Oliveira-Castro & Loewenstein,1971). Moreover, rise in cytoplasmic Ca2] may causerise in cytoplasmic H] because of shared cytoplasmicbuffers. Given the probable lack of homology betweenconnexins and gap junctions in the arthropod line(Barnes, 1994), the old data are of limited predictivevalue for connexins in any case. Although it is likelythat rises in Ca2] do decrease conductance of connexinchannels, there is still no good dose response curve orevidence of direct versus indirect action. The newmethods of single channel recording will allow, hope-fully, resolution of this question. What is (and was)clear is that low levels of Ca2] will permeate gapjunctions (Saez et al., 1989), and waves of increase inintracellular Ca2] have been observed to propagatebetween many kinds of coupled cells (Dani & Smith,1995), although in some instances IP3 rather than Ca2]

may be the signal that propagates between cells (Sand-erson, 1995). In any case the low levels of cytoplasmicfree Ca2] that healthy cells permit may never be highenough to close gap junctions, but in dying cells rise inCa2] is likely to be a factor in uncoupling them fromhealthy neighbours.

After Turin and Warner (1977) showed uncouplingin amphibian blastulae by treatment with weak acids(which cross the membrane in undissociated form andthen dissociate to acidify the interior), we confirmedthat cytoplasmic acidification decreases junctionalconductance in a variety of cell types and using in-tracellular electrodes were able to obtain reasonabletitration curves (Spray et al., 1981b; Bennett et al., 1988).Onset of block and recovery can be produced in tens ofseconds. Application of CO2 or other weak acid wasfor some time the best way to decrease junctionalconductance between cells. As one would expect justfrom the existence of the connexin gene family, not alljunctions are equally sensitive, but none appear to becompletely insensitive. Now Delmar and colleaguesare defining sites in the C]43 molecule responsiblefor the H] effect on conductance, which appears to bedistinct from voltage gating (Ek et al., 1994), althoughas noted above H] affects voltage sensitivity in am-phibian blastomers (which express Cx38; Bennettet al., 1988). The action of H] does not appear to bea normal way of controlling junctional conductance,but coupling may be affected by rises in acidity duringpathological conditions such as anoxia, ischemia andseizures.

After uncoupling by H] came uncoupling with longchain alcohols, n-heptanol and n-octanol ( Johnston

et al., 1980), and still more recently halothane (Burt& Spray, 1989). These agents, like Ca2] and H], alsoappear quite non-specific in the junctions on whichthey act and are potent in both vertebrate and arthro-pod junctions. These agents have provided the bestmethod of reducing junctional conductance to deter-mine that coupling is gap junction mediated, to estab-lish the effect of loss of coupling or to discriminatesingle channel currents. The major problem with theseagents, as with H], is action on other cell properties;many of these treatments tend to block excitability, forexample, which greatly limits their usefulness withrespect to excitable cells such as neurons. Newer ap-proaches dependent on molecular biology offer morespecificity. Antibodies to extracellular loops can blockjunction formation (Meyer et al., 1992), connexin ex-pression can be reduced by treatment with antisenseoligonucleotides (Moore & Burt, 1994) and connexinscan be knocked out by homologous recombination(Reaume et al., 1995; Nelles et al., 1996).

Voltage dependence of junctional conductance

In 1980 another form of modulation of vertebrate gapjunctions was discovered. Dave Spray, Andy Harrisand I observed that the junctional conductance be-tween amphibian blastomeres was strongly depen-dent on transjunctional voltage (Harris et al., 1981).Again, Anne Warner’s laboratory had made an initialsuggestive observation (Blackshaw & Warner, 1976).Previously, certain electrical synapses in the hatchet-fish had been found to rectify, i.e., their conductancewas increased by one sign of transjunctional voltageand decreased by the opposite sign (Auerbach & Be-nnett, 1969b; Hall et al., 1985); the rectification tendedto make the transmission unidirectional as in the rec-tifying synapses of crayfish (Furshpan & Potter, 1959).When we voltage clamped pairs of blastomeres wefound that transjunctional voltage, Vj, of either signdecreased junctional conductance, gj (Fig. 10A). (gj waslittle affected by the voltage between the interior of thecells and the outside, Vi–o, as is commonly true ofconnexins, but untrue of some invertebrate gap junc-tions (Verselis et al., 1991).) The symmetry of the gj/Vj

relation is consistent with the symmetry of the coupledcells. The steady state conductance for one polarity oftransjunctional voltage was well fit by a Boltzmannrelation (Fig. 10B), which suggests a voltage gate thatcan be in either of two states with the energy differ-ence between them linearly dependent on voltage (butBoltzmann relations can give a reasonable fit to almostany sigmoid and the predictive value is not great).We hypothesized that there were two gates, one ineach hemichannel, one closed by one polarity of Vj, theother closed by the opposite polarity (Fig. 11). Becausegj was little affected by Vi–o, we placed the gates at thecytoplasmic ends of the channel. Actually, we did not


Fig. 10. Voltage dependence of junctional conductance inamphibian blastomeres. (A) Records from a dual voltageclamp experiment on a coupled cell pair. One cell wasstepped to a new voltage, while the second cell was heldconstant. The current applied in the second cell representsthe transjunctional current flowing as a result of the voltagestep and is displayed here upward for positive Vj. For smallvoltages of either sign, the current decreases only slightlyduring the step. For larger voltages of either sign, the currentrelaxes exponentially to a lower value. (B) Steady-state junc-tional conductance, gj, as a function of transjunctional volt-age, Vj . gj decreases steeply for Vj of either sign. The smoothcurves are Boltzmann relations for positive and negativeVj as would be observed if the energy difference betweenopen and closed states were a linear function of transjunc-tional voltage. The parameters of the particular curve are forthe equivalent of about 6 electron charges moving throughthe entire transjunctional voltage. (C) Effects of currentpulses. During large enough current pulses of either sign,the voltage in the directly polarized cell increases in a regen-erative manner while the voltage in the other cell declines.The cells change from a well-coupled to a poorly-coupledstate as a result of voltage dependence of the junctionalconductance. (D) Bistability of coupling resulting from volt-age dependence of gj and difference in resting potentialsbetween two cells of a pair. A train of pulses in one cell (V

2)

leads to progressive decrease in junctional conductance untilthe cells uncouple, the pulsed cell having the more negativeresting potential. The break in the record represents 100 s.Near the end of the record an oppositely direct pulse wasapplied that reduced the transjunctional voltage and al-lowed the cells to recouple. From Harris and colleagues,1983 and Spray and colleagues, 1981a.

know which gate was closed by which polarity ofVj until Vytas Verselis injected acid into one of apair of coupled cells (Bennett et al., 1988); this proced-ure modified gating properties for one polarity ofvoltage, and we concluded that the effect was onthe gate on the acidified side. Our heuristic diagram(Fig. 11) turned out to be right for these junctions;

transjunctional voltage closes the gate on the positiveside. We were, and are, uncertain why junctions be-tween amphibian blastomeres have this voltage sensi-tivity, which we observed in a urodele, several ranidsand Xenopus. (It also occurs in ascidian blastomeres,but not in teleost blastomeres; Knier et al., 1986.) Thedegree of sensitivity is comparable to that of Na chan-nels, although the transitions are much slower. We didshow that if there were modest resting potential differ-ences between cells, the voltage dependence couldlead to bistability in which the cells existed in stablecoupled or uncoupled conditions depending on priorhistory (Fig. 10D) (Harris et al., 1983). We thought thatthis property might be involved in setting up embry-onic compartments.

The study of voltage dependence has come a longway from our initial work. Cloned connexins can beexpressed in Xenopus oocytes or communication defi-cient cell lines for determination of macroscopic andsingle channel properties, respectively. All vertebrateconnexins tested have some degree of voltage depend-ence (except Cx33 which apparently does not formfunctional junctions and interferes with formation ofjunctions by Cx37 when the two are expressed to-gether; Chang et al., 1994). For some, e.g., Cx43, thedependence is weak, and in heart muscle, constancy oftransmission in the face of depolarization is likely to befunctional. Conversely Cx37 is quite voltage sensitive(Reed et al., 1993) and is found in neural progenitorcells (R. Rozental, personal communication) as well asskin (Goliger & Paul, 1994). This sensitivity might giverise to modulation at electrical or dual electrical andchemical synapses. Hemichannels formed of differentconnexins can close in response to opposite polaritiesof transjunctional voltage (Verselis et al., 1994). Whenhemichannels of opposite polarity form junctions,their channels respond to the same polarity of Vj,giving rise to strongly rectifying gj/Vj relations (Barrioet al., 1991; Verselis et al., 1994). These observationssuggest that rectifying synapses arise because thepre- and postsynaptic cells express connexins withopposite polarities of voltage dependence. Structurefunction studies are revealing components of thevoltage gating mechanism (Verselis et al., 1994). Re-markably, a charge just one or two positions fromthe N-terminus, which is cytoplasmic, is part of thegating charge, and changing this charge by site di-rected mutagenesis can reverse the polarity of gatingof several connexins (Verselis et al., 1994; unpub-lished findings). Another charge change at the borderbetween the first membrane spanning and first extra-cellular loop domains can also reverse polarity ofgating or alternatively increase its voltage sensitivity.The changes are consistent with movement of bothof these charges towards the cytoplasm. Thus, thevoltage sensor appears to extend quite far withinthe molecule.

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Fig. 11. Diagram of a gap junction channel with voltage gates at either end. Transjunctional voltage, Vj , closes the gate in thehemichannel on the relatively positive side, giving a symmetrical relation between junctional conductance, gj, and Vj.Heterotypic channels comprised of hemichannels with different voltage sensitivity have asymmetric gj/Vj relations. Rectifica-tion is observed if the hemichannels have opposite gating polarities, so that both are closed by the same polarity of Vj whenthey form a heterotypic channel.

At least seven connexins are expressed in rodentnervous system, Cx26, Cx32, Cx37, Cx40, Cx43, Cx45and Cx46; how cells expressing one or more ofthese connexins interact with other cells expressingthe same or different connexins is being determinedwith molecular and electrophysiological techniques.Some connexins are compatible in that they form func-tional heterotypic junctions, which may be quite sym-metrical or very rectifying. Other connexins areincompatible and will not form functional junctionsand may even interfere with junction formation byotherwise compatible connexins (Chang et al., 1994;Elfgang et al., 1995; White et al., 1995b). Junctionalproperties are quite variable and are usually, but notalways, predictable from the properties of their consti-tutive connexins in other combinations. Single channelrecording is illuminating aspects of the gating mecha-nism; many but not all of these aspects would havebeen predicted from macroscopic properties of thejunctions.

There have been several surprises. Gap junctionsmay be more permselective than previously believed,e.g., Cx45 channels are cation selective (Veenstraet al., 1994). Moreover, Cx46 can open as an isolatedhemichannel without an apposed hemichannel, inwhich state it is quite cation selective, and the voltage

sensitivity of opening of the isolated hemichannel iswithin the physiological range of membrane potentialchanges (Trexler et al., 1996). It remains to be deter-mined whether these hemichannels ever do openunder physiological conditions. The permeability torelatively large ions could lead to deleterious effects,and it has generally been thought that hemichannels inthe surface membrane not in junctions are closed. Theresidual conductance of gap junctions in the face ofstrong transjunctional voltage is, in Cx43 and Cx46at least, due to a conductance substate induced at thesevoltages (Moreno et al., 1994; Perez-Armendariz et al.,1994; Trexler et al., 1996). When channels first form, theinitial opening from zero conductance is slow com-pared to gating transitions of formed junctions, mostof which are to a substate; complete closures are slowlike the openings from the completely closed state(Bukauskas et al., 1995). Similar slow transitions werereported in the first single channel recordings of gapjunction gating (Neyton & Trautmann, 1985). They arealso seen with Cx46 hemichannels and can be ex-plained as unresolved transitions between multiplesubstates associated with opening of the extracellularloops (Trexler et al., 1996).

In situ hybridization and sequence specific anti-bodies will reveal which connexins are expressed in


the nervous system and where. Application of patchclamping (and probably optical methods) to disso-ciated cells and brain slices in culture will provideevidence as to the actual function of the gap junctionsat these synapses. Specific antibodies to block forma-tion, antisense techniques to reduce connexin ex-pression and knock-out homologous recombinationshould provide important adjuncts to the directmeasurement of electrical coupling.

Acknowledgements

This work depended on numerous colleagues most ofwhose names can be found in the reference list; I amdeeply indebted to them. Funding came from manyNIH and NSF grants over the years, initially toHarry Grundfest. A major support for 26 years hasbeen NS-07512. I am the Sylvia and Robert S. Olnick,Professor of Neuroscience.

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gap junctions as electrical synapses

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