molecular and functional asymmetry at a vertebrate ...€¦ · molecular and functional asymmetry...

Neuron

Article

Molecular and Functional Asymmetryat a Vertebrate Electrical SynapseJohn E. Rash,1 Sebastian Curti,2,3 Kimberly G. Vanderpool,1 Naomi Kamasawa,4 Srikant Nannapaneni,2

Nicolas Palacios-Prado,2 Carmen E. Flores,2 Thomas Yasumura,1 John O’Brien,5 Bruce D. Lynn,6 Feliksas F. Bukauskas,2

James I. Nagy,6 and Alberto E. Pereda2,*1Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA2Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA3Laboratorio de Neurofisiologıa Celular, Departamento de Fisiologıa, Facultad de Medicina, Universidad de la Republica,

Montevideo 11800, Uruguay4Max Planck Florida Institute, Jupiter, FL 33458, USA5University of Texas Health Science Center, Houston, TX 77030, USA6Department of Physiology, University of Manitoba, Winnipeg, MB R3E 0J9, Canada

*Correspondence: [email protected]

http://dx.doi.org/10.1016/j.neuron.2013.06.037

SUMMARY

Electrical synapses are abundant in the vertebratebrain, but their functional and molecular complex-ities are still poorly understood. We report herethat electrical synapses between auditory afferentsand goldfish Mauthner cells are constructed byapposition of hemichannels formed by two homo-logs of mammalian connexin 36 (Cx36) and that,while Cx35 is restricted to presynaptic hemiplaques,Cx34.7 is restricted to postsynaptic hemiplaques,forming heterotypic junctions. This molecular asym-metry is associated with rectification of electricaltransmission that may act to promote cooperativitybetween auditory afferents. Our data suggest that,in similarity to pre- and postsynaptic sites at chem-ical synapses, one side in electrical synapsesshould not necessarily be considered the mirrorimage of the other. While asymmetry based on thepresence of two Cx36 homologs is restricted toteleost fish, it might also be based on differencesin posttranslational modifications of individual con-nexins or in the complement of gap junction-associ-ated proteins.

INTRODUCTION

While the physiological importance of electrical synaptic trans-

mission in cold-blooded vertebrates has long been established

(Bennett, 1977), progress over the last decade has also revealed

the widespread distribution of electrical synapses, and this

modality of synaptic transmission was reported to underlie

important functional processes in diverse regions of the

mammalian CNS (Connors and Long, 2004). Consequently, elec-

trical transmission is now considered an essential form of inter-

neuronal communication that, together with chemical transmis-

sion, dynamically distributes the processing of information

within neural networks. In contrast to detailed knowledge of

the mechanisms underlying chemical transmission, far less is

known about how the molecular architecture or the potentially

diverse biophysical properties of electrical synapses encoun-

tered in physiologically disparate neural systems govern their

function or impact on characteristics of electrical transmission

in those systems.

Electrical synaptic transmission is mediated by clusters of

intercellular channels that are assembled as gap junctions

(GJs). Each intercellular channel is formed by the docking of

two hexameric connexin hemichannels (or connexons), which

are individually contributed by each of the adjoining cells,

forming molecular pathways for the direct transfer of signaling

molecules and for the spread of electrical currents between

cells. As a result, electrical synapses are often perceived as

symmetrical structures, at which pre- and postsynaptic sites

are viewed as the mirror image of each other. Connexons

are formed by proteins called connexins that are the products

of a multigene family that is unique to chordates (Cruciani

and Mikalsen, 2007). Because of its widespread expression

in neurons, connexin 36 (Cx36) is considered the main

‘‘synaptic’’ connexin in mammals. In contrast to other connex-

ins, such as some found in glia (Yum et al., 2007; Orthmann-

Murphy et al., 2007), all pairing configurations tested so far

indicate that Cx36 forms only ‘‘homotypic’’ intercellular chan-

nels (Teubner et al., 2000; Li et al., 2004), where connexons

composed of Cx36 pair only with apposing Cx36-containing

connexons. Notably, the number of neuronal connexins is

higher in teleost fishes, which, as a result of a genome duplica-

tion (Volff, 2005), have more than one homolog gene for most

mammalian connexins (Eastman et al., 2006). This raised the

possibility of more complex configurations of neuronal con-

nexin coupling in teleost fish evolving in response to functional

demands.

Because of their experimental access, auditory ‘‘mixed’’

(electrical and chemical) synapses on the teleost Mauthner cell

(M-cell) (a reticulospinal neuron involved in tail-flip escape

responses; Faber and Pereda, 2011), known as ‘‘large myelin-

ated club endings’’ (CEs), constitute a valuable model for

studying vertebrate electrical transmission (Pereda et al.,

Neuron 79, 957–969, September 4, 2013 ª2013 Elsevier Inc. 957

mailto:[email protected]


http://crossmark.dyndns.org/dialog/?doi=10.1016/j.neuron.2013.06.037&domain=pdf

Figure 1. Presence of Two Homologs of Cx36 at CEs

(A) Diagram of the M-cell. Auditory afferents terminate as CEs in the distal

portion of the lateral dendrite, formingmixed (electrical plus chemical) synaptic

contacts (dashed box).

(B–D) Laser scanning confocal projection of the distal portion of the lateral

dendrite. Double immunolabeling with a monoclonal Cx35 antibody (C, red)

and a polyclonal Cx34.7 (IL) antibody (D, green) shows a high degree of

colocalization at individual CEs, shown by red/green overlay (B, asterisks).

(E–G) Highmagnification of an individual CE showing intense punctate labeling

for Cx35 (F, red) and Cx34.7 (G, green) and high colocalization (E).

(H) Confocal line-scan imaging (blue line in inset) illustrates the high degree of

colocalization of Cx35 and Cx34.7.

(I) Plot represents the ratio of superposition of Cx35 to Cx34.7 labeling at

individual CEs (converted to percentage) against the ratio of Cx34.7 to Cx35

(n = 30).

See also Figures S1 and S2.

Neuron

Molecular Asymmetry and Electrical Rectification

958 Neuron 79, 957–969, September 4, 2013 ª2013 Elsevier Inc.

2004). We previously reported that connexin 35 (Cx35) (O’Brien

et al., 1998), a fish homolog of mammalian neuronal Cx36

(Condorelli et al., 1998), is present at CEs (Pereda et al., 2003).

Given the genome duplication that occurred in teleost fish, we

investigated the presence of an additional Cx36 homolog at

these terminals. Using immunofluorescence and ultrastructural

approaches, we show that a second homolog of Cx36, connexin

34.7 (Cx34.7) (O’Brien et al., 1998), is also present at CEs.

Strikingly, matched double-replica freeze-fracture immunogold

labeling revealed that Cx35 is restricted to presynaptic CE

hemiplaques, whereas Cx34.7 is restricted to postsynaptic

M-cell hemiplaques. Asymmetry in the molecular composition

of adjoining connexons was proposed to allow electrical

rectification at some GJs (Barrio et al., 1991; Phelan et al.,

2008). Consistent with this notion, our estimates of GJ resistance

in each direction revealed a near 4-fold difference in con-

ductance, favoring the spread of postsynaptic membrane

responses to presynaptic endings, which, by acting as a

mechanism of lateral excitation (Pereda et al., 1995; Curti and

Pereda, 2004), would facilitate the fish’s escape. Thus, molecu-

lar asymmetries in neuronal gap junctions can underlie complex

functional properties and suggest that the apposed sides of

electrical synapses are not necessarily the mirror images of

each other.

RESULTS

Cx35 and Cx34.7 Colocalize in Club EndingsThe large size and distinctive morphology of the M-cell (Fig-

ure 1A) allows the imaging of long stretches of membrane in a

single optical section. Because of their unusually large size,

CEs can be unequivocally identified on the distal portion of the

M-cell lateral dendrite using Cx35 labeling (Figures 1B and 1C;

Pereda et al., 2003; Flores et al., 2008). To determine whether

other teleost homologs of Cx36 are present at CEs, we investi-

gated whether Cx35 colocalizes with Cx34.7 at CEs by perform-

ing double immunofluorescence labeling using an anti-Cx35

antibody (Chemicon MAB3043) and an anti-Cx34.7 intracellular

loop (IL) antibody (see below; Experimental Procedures; Table

S1 available online). Both Cx35 and Cx34.7 antibodies showed

intense punctate staining and colocalization at contacts

between CEs and M-cells (Figures 1B–1D). We previously

showed that the number of anti-Cx35 fluorescent puncta at indi-

vidual CEs (Figures 1E–1G) was consistent with ultrastructural

demonstration of 63–243 closely spaced GJ plaques at these

terminals (Tuttle et al., 1986), suggesting that each punctum

represents an individual plaque (Flores et al., 2008). Accordingly,

confocal line-scan imaging illustrated the colocalization of label-

ing for both antibodies at single puncta, suggesting that both

connexins coexist at individual plaques (Figure 1H). We quanti-

fied the colocalization of Cx35 with Cx34.7 (and vice versa) using

confocal reconstruction of individual terminals, as identified by

shape and Cx35 labeling (Figure 1F). Averaged over individual

endings, 85.04% (±9.12 SD) of the area of Cx35 immunolabeling

also showed Cx34.7 labeling and 81.23% (±8.34 SD) of the area

of Cx34.7 labeling showed Cx35 labeling (n = 30) (Figure 1I).

Thus, although not completely overlapping, the two proteins

exhibit a high degree of colocalization in CEs.

Neuron


Ultrastructural Analysis Reveals that Cx35 and Cx34.7Are Differentially Segregated to Pre- versusPostsynaptic SidesTo confirm that Cx35 and Cx34.7 colocalize at individual GJ

plaques, we performed conventional freeze-fracture replica

immunogold labeling (FRIL), which allows broad expanses of tis-

sues to be examined and facilitates unambiguous assignment of

specific connexin labeling to GJ hemiplaques in either of two

apposed cells (see Supplemental Experimental Procedures).

Four replicas of goldfish hindbrain contained CE synapses on

identified M-cells. The CE terminals were identified on confocal

grid-mapped M-cells that had been injected with Lucifer yellow

during in vivo recordings prior to tissue fixation as well as in one

set of matched double replicas prepared by SDS-FRIL (see

Supplemental Experimental Procedures). Samples were either

single-labeled with anti-Cx36 Ab298, which binds to both

Cx34.7 and Cx35 (see below), or double-labeled for Cx35 and

Cx34.7 IL. In a double-labeled replica of a positively identified

M-cell, labeling for Cx35 was found directly associated with GJ

plaques in presynaptic membranes of CEs (n = 20 GJs). In

contrast, labeling for Cx34.7 was only on identified M-cell post-

synaptic membranes (n = 53 GJs). Consistent with this distribu-

tion, anti-Cx36 Ab298, which recognizes both Cx35 and Cx34.7

(see next section and Table S1), was found to label both pre-

and postsynaptic membranes (data not shown, but see data in

Pereda et al., 2003). Such differential distribution to pre- versus

postsynaptic membranes was investigated further by double-

immunolabeling for Cx35 and Cx34.7 using matched double-

replica FRIL (DR-FRIL). Initially, a sample prepared for DR-FRIL

was fractured andmajor portions of bothmatching complements

were retrieved and labeled. In one of the two M-cell comple-

ments, more than 400 labeled GJs were found; 367 were viewed

toward theM-cell side of the junction (Figures 2A–2D), all ofwhich

were labeled for Cx34.7 and none for Cx35; and 79 were viewed

from the M-cell side of the synapse toward the CE (Figure 2E), all

of whichwere labeled for Cx35 and none for Cx34.7. A diagramof

that same cell is indicated in Figures 2F and 2G, illustrating the

two primary views seen in Figures 2D and 2E.

To further investigate this apparent GJ connexin asymmetry,

analysis was performed on matching complements of individual

M-cell/CE GJs in these same samples. However, because of

damage to one of the matching replicas, only about 30 M-cell/

CE GJs could be matched in the two complementary replicas

(Figure 3). Of those 30 matching complements, 100% had label-

ing for Cx35 (10 nm gold beads) within the CE plasma mem-

brane, without labeling for Cx34.7 IL, and 100% had labeling

for Cx34.7 IL (5 nm gold beads) within the postsynaptic M-cell

plasma membrane, with no labeling for Cx35. Thus, whether

examined in single replicas or in matched complementary

double replicas of the same GJ hemiplaques, Cx35 was

restricted to the CE side of GJs (presynaptic hemiplaques) and

Cx34.7 was present only in the M-cell side of GJs (postsynaptic

hemiplaques), unambiguously demonstrating that GJ channels

between CEs and the M-cell dendrite are heterotypic.

Specificity of Anti-Connexin AntibodiesBecause of substantial amino acid sequence identity of Cx35

and Cx34.7, the specificity of the antibodies used here is critical

for the accurate identification of these two connexin homologs.

Our previous studies on connexins at CEs focused largely on

Cx35 at these synapses, using either anti-Cx35 antibodies or

anti-Cx36 antibodies that were shown to recognize Cx35. In

the present study, HeLa cells transfected with Cx34.7 or Cx35

were used to confirm the quality and specificity of a set of anti-

Cx34.7 antibodies and to establish which of the previously

utilized as well as currently available anti-Cx36 antibodies either

do or do not cross-react with Cx34.7 or Cx35 (Table S1).

HeLa cells were found to readily express Cx34.7 upon trans-

fection, and robust immunofluorescence detection of this con-

nexin both intracellularly and at plasma membrane contacts

was obtained with anti-Cx34.7 IL (Figure S1A1). The same cul-

ture labeled with anti-Cx36 Ab39-4200 showed codetection

and subcellular colocalization of labeling (Figures S1A2 and

S1A3), indicating Ab39-4200 recognition of Cx34.7 and therefore

serving as a positive control for Cx34.7 expression. The anti-

Cx36 Ab298 previously shown in our earlier study to recognize

Cx35 (Pereda et al., 2003) also recognized Cx34.7 (Figure S1B1)

and produced labeling that corresponded with labeling pro-

duced by Ab39-4200 (Figures S1B2 and S1B3). We next tested

immunofluorescence detectability of Cx35 with anti-Cx34.7 IL

in HeLa cells transfected with Cx35-enhanced yellow fluores-

cent protein (eYFP). Clusters of HeLa cells with high transfection

efficiency displayed intense intracellular eYFP fluorescence as

well as detection of Cx35-eYFP at cell-cell contacts (Figures

S1C1 and S1E1). In these cultures, Cx35 was not recognized

by anti-Cx34.7 IL (Figures S1C2 and S1C3), indicating specificity

of this antibody for Cx34.7. In contrast, while Cx34.7-transfected

cells showed robust labeling of Cx34.7 with anti-Cx36 Ab39-

4200 (Figure S1D1), polyclonal anti-Cx36 Ab51-6300 did not

cross-react with Cx34.7 in this same culture (Figures S1D2 and

S1D3) but showed robust detection of Cx35 (Figures S1E2 and

S1E3). See Figure S2 for additional antibodies tested. In addi-

tion, we previously established that anti-Cx35 (Chemicon

MAB3043) antibody does not crossreact with Cx34.7 (Pereda

et al., 2003).

Electrical Transmission between CEs and the M-cell IsAsymmetricHeterotypic GJ channels have been associated with asymmetry

of electrical transmission (Barrio et al., 1991; Phelan et al., 2008).

While simultaneously recording a single CE afferent at the VIIIth

nerve root and the M-cell lateral dendrite (Figure 4A), we found

a dramatic difference between orthodromic and antidromic

coupling coefficients (CCs), calculated using the M-cell and CE

action potentials and their respective coupling potentials (CC =

coupling/action potential). The CCs averaged 0.009 ± 0.001

(SEM) in the orthodromic direction and 0.083 ± 0.009 (SEM) in

the antidromic direction (p < 0.0005; n = 36). The �9-fold

disparity indicates that electrical transmission is stronger in the

antidromic direction. This difference is observed in the simulta-

neous recording illustrated in Figure 4A and is more clearly

observed in the experiment of Figure S3A, where multiple CEs

terminating in the same lateral dendrite were recorded sequen-

tially while maintaining the dendritic recording electrode. There

was a dramatic difference for CCs in the antidromic direction

at eachCE (Figure S3B), indicating that the functional asymmetry


Figure 2. Pre- versus Postsynaptic Con-

nexins

Low- to high-magnification images of a single

M-cell after double labeling with 5 nm gold beads

for Cx34.7 (using antibody 2930-2 IL) and with

10 nm gold beads for Cx35 using MAB3045.

(A–C) M-cell lateral dendrite (A, yellow overlay)

surrounded by CEs (pale green overlays) and by

additional unidentified axon terminals (not

colored). Boxed area is shown at higher magnifi-

cation in (B), and the boxed area in (B) is shown at

higher magnification in (C).

(D) High magnification of boxed area in (C),

showing view from CE E-face (green overlay)

toward the M-cell P-face (yellow overlay), with

connexins in the M-cell plasma membrane labeled

only for Cx34.7 (5 nm gold beads), whether as

M-cell P-face connexons (blue overlay) or CE

E-face pits (blue-green overlay). Two 10 nm gold

beads that are not on a gap junction represent

nonspecific background or ‘‘noise’’ (indicated by

the ‘‘barred circle’’). Purple overlays are clusters of

P-face pits representing imprints of glutamate

receptors. (See glutamate receptor E-face IMPs

in E.)

(E) View from the M-cell E-face (yellow overlay)

toward the CE P-face (green overlay) at the

opposite end of the same obliquely fractured

M-cell as shown in (A)–(D). The postsynaptic

membrane contains clusters of 10 nm E-face

intramembrane particles (IMPs; purple overlays)

corresponding to glutamate receptor particles

(Tuttle et al., 1986; Pereda et al., 2003). After SDS

washing, connexons remaining in the CE plasma

membrane, beneath replicated CE P-face IMPs

(green overlay) and M-cell E-face pits (yellow-

green overlay), are labeled solely for Cx35 (10 nm

gold beads).

(F and G) Stylized diagrams (i.e., molecular versus

cellular components not to same scale) illustrating

the location of the fracture plane coursing diago-

nally through a M-cell (F, yellow). The resulting

labeled replica (diagrammed in G) illustrates that

connexons remain in the cell whose residual

cytoplasm is retained beneath the replica. The

apparent labeling of gap junction E-face pits

(which are devoid of connexin proteins and

therefore cannot be labeled) occurs because

intact but unreplicated connexons remain strongly

adsorbed to the platinum/carbon replica at the bottom of the E-face pits, where they remain for subsequent labeling (Fujimoto, 1995). Clusters of 10 nmglutamate

receptor IMPs are retained in the extraplasmic leaflet, providing a marker for recognizing the M-cell plasma membrane E-face, as previously demonstrated

(Pereda et al., 2003). Lavender circles are synaptic vesicles. Small black dots are gold beads for Cx34.7; larger black dots are gold beads for Cx35.


Neuron


represents a general property of CEs likely operating under

physiological conditions, as it was observed using physiological

signals, such as action potentials.

Electrical Synapses between CEs and theM-cell RectifyThe strength of electrical transmission (amplitude of the coupling

potential) does not solely depend on the conductance of the GJ

channels but also on the passive properties determined by the

resistance (and capacitance under some conditions) of the

coupled neurons. The relatively smaller size of CEs indicates


that their input resistance is likely higher than that of the M-cell

dendrite, thus contributing to the asymmetry between ortho-

dromic and antidromic CCs. To evaluate the contribution of

heterotypic GJ channels to asymmetric electrical transmission,

we investigated possible asymmetries in GJ resistance between

CEs and the M-cell. Rectification refers to the propensity of

some electrical synapses to display differential resistance to

current flow in one versus the other direction across the junction

between two coupled cells (Furshpan and Potter, 1959). While

properties of junctional conductance (inverse of resistance) are

Figure 3. Connexin Localization by DR-

SDS-FRIL

Matched double replicas of twoGJs from the same

replica as in Figure 2, with diagrams illustrating

labeling of the matched hemiplaques.

(A and B) Matched GJ hemiplaques viewed toward

the M-cell plasma membrane P-face (A, yellow

overlay), with 5 nm gold beads (small arrows) for

Cx34.7 present beneath both P-face IMPs (blue

overlay) of the M-cell and beneath E-face pits

(blue-green overlay) of the CE plasma membrane

(green overlay). (B) View toward the CE (light green

overlay), with 10 nm gold beads (large arrows)

labeling for Cx35 beneath E-face pits (yellow-

green overlay) of the extraplasmic leaflet of the

M-cell (yellow overlay) and beneath P-face con-

nexon IMPs (light green overlay) of the CE plasma

membrane (light green overlay). These matched

complementary replicas document that CE/M-cell

GJs are heterotypic/asymmetric.

(C) Diagram of the fracture plane through a gap

junction between a CE (left panel, top) and the

M-cell (left panel, bottom). The fracturing process

(middle panel) separates all connexons (blue in

M-cell; green in CE) at their points of contact in the

extracellular space, with all connexons of the CE

remaining with the upper tissue fragment and all

connexons of the M-cell remaining with the lower

fragment. Circular arrow and curved arrow indicate that the upper tissue fragment is inverted for labeling (right panel). Center panel reveals 5 nm gold beads

labeling only Cx34.7 in the M-cell cytoplasm, regardless of whether E-face pits of the CE or P-face IMPs of the M-cell are replicated. Right panel reveals 10 nm

gold beads labeling only Cx35 in the CE cytoplasm, whether beneath M-cell E-face pits or beneath CE P-face IMPs.


Neuron


generally examined with simultaneous recordings from two cells

under voltage clamp configuration (Barrio et al., 1991), this

approach in our case would require simultaneous in vivo intrater-

minal and intradendritic recording, which is feasible (Pereda

et al., 2003) but not sufficiently stable for analysis of rectification.

Moreover, the resistance of the presynaptic electrode and

geometrical characteristic of the afferents make it impractical

to use the voltage clamp configuration to directly determine

junctional resistance. Therefore, we followed an established indi-

rect approach (Bennett, 1966; Devor and Yarom, 2002), where

junctional resistance can be estimated from measurements of

CCs in each direction (from pre- to post and vice versa), in com-

bination with measurements of the input resistances of the

coupled cells (see Experimental Procedures). While this

approach might also be challenging in some cell types (GJs

are dendrodendritic in most mammalian neurons), the M-cell

and the CEs offer several unusual anatomical and physiological

characteristics that make it possible to estimate these parame-

ters in vivo: (1) CE afferents terminate with a single contact and

are tightly segregated to the distal portion of the lateral dendrite

of the M-cell; (2) the M-cell lateral dendrite as well as both the

axons and terminals of CEs are accessible for intracellular re-

cordings; and (3) the M-cell and the CEs have comparable and

unusually fast membrane time constants, estimated to be

400 ms in the M-cell (Fukami et al., 1965) and 200 ms in CEs (Curti

et al., 2008), which allow the use of physiological signals, such as

action potentials, for measurements of CCs. Due to spatial con-

siderations, measurements of CCs during simultaneous record-

ings of CE afferents in the VIIIth nerve root and theM-cell dendrite

are useful to expose asymmetry of electrical transmission (Fig-

ures 4A and S3B) but not accurate enough for estimating GJ

conductance (see below). To overcome this problem, we calcu-

lated average values of CCs for the population of afferents, using

values obtained under various experimental arrangements that

maximize their accuracy (see below).

The ‘‘population CC’’ in the orthodromic direction (CE to

M-cell) for a number of CEs was estimated as the ratio between

the average amplitude of the electrical component (or coupling

potential) of the unitary postsynaptic potential and the average

amplitude of the presynaptic spike (CC, postsynaptic coupling

potential/presynaptic spike; Figure 4A). The orthodromic

coupling potential (recorded during paired recordings with intra-

dendritic recordings in the terminal field of CEs) averaged 0.73 ±

0.04 mV SEM (n = 76). (Because the strength of electrical synap-

ses between individual CEs varies dramatically [Smith and

Pereda, 2003], it was not possible to assign differences in the

amplitude of individual coupling potentials to their relative posi-

tion within the dendritic field and therefore correct for potential

electrotonic attenuation. Thus, although potentially slightly

underestimated, we believe the average amplitude of ortho-

dromic coupling potentials represents the most appropriate

value to use for calculating the CC in the orthodromic direction.)

During simultaneous recordings, the amplitude of the presynap-

tic spike evoked at the recording site with long (200 ms)

depolarizing pulses does not represent the spike that ultimately

generates coupling, as the spike recorded at the site of depolar-

ization regenerates in subsequent nodes and, finally, at the

presynaptic terminal (see Figure S4). More importantly, its


Figure 4. Electrophysiological Recordings

for Estimates of Junctional Conductance

(A) Experimental arrangement. AD and pre- and

postsynaptic electrodes are indicated.

(B) Simultaneous pre- and postsynaptic re-

cordings between CEs and the M-cell lateral

dendrite allow measuring orthodromic and anti-

dromic coupling potentials in the same terminal.

Here and elsewhere, unless indicated, traces

represent the average of at least ten single re-

sponses. Left panel: Estimating dendritic Rn.

Excitatory postsynaptic currents (EPSC) evoked

by extracellular stimulation of the posterior VIIIth

nerve (population response) obtained while

clamping at resting potential (�76 mV) using

single electrode voltage clamp (SEVC). Top

right: Currents evoked by command pulses

of +10, +5,�15, and�25mV from resting potential

(arrowhead indicates the time at which currents

were measured). Bottom right: voltage-current

relationship plotted for a larger range of command

pulses. Conductance (G) was calculated from the

slopes of the fitted line.

(C) Intraterminal recordings (club ending) obtained

after an initial recording of the antidromic spike (AD

spike) in the M-cell lateral dendrite (M-cell, left),

showing antidromic coupling (center). Recordings

are also illustrated scaled (right) (modified from

Curti and Pereda, 2004; Smith and Pereda, 2003).

(D and E) Spatially controlled sequential intra-

dendritic recordings of the AD spike obtained after

an initial extracellular field recording of the AD

spike in the M-cell axon cap (B).

(F) Graph plots the decay of the AD spike with

distance. Data were fit with a single exponential

(r2 = 0.99). Orange bar indicates the location of the

CEs terminal field in the lateral dendrite.

See also Figure S4.

Neuron


amplitude is affected by the pulse depolarization. Therefore, for

values of presynaptic spike amplitude, we used short depolariz-

ing pulses, at which spikes initiate from resting potential and are

likely representative of those normally occurring at the contact,

averaging 87.6 ± 0.9 mV SEM (n = 203). These measurements

yielded an orthodromic CC of 0.008. The input resistance of

the M-cell lateral dendrite was directly measured under single-

electrode voltage-clamp configuration during intradendritic re-

cordings (see Experimental Procedures) and found to be, on

average, 1.32 ± 0.3 MU SEM (n = 9; Figure 5B).

The population antidromic CC (M-cell to CE) was calculated as

the ratio between the amplitude of the antidromic (AD) coupling

potential (the coupling of the antidromic spike of the M-cell in the

CE) and the amplitude of the antidromic M-cell spike (AD spike)


recorded in the dendrite (Figure 5C;

CC, AD coupling potential/AD spike).

Because the AD coupling potential is

greatly reduced by electrotonic attenua-

tion when recorded at the VIIIth nerve

root during simultaneous recordings, we

estimated its average value by performing

intraterminal recordings in the vicinity of

the M-cell lateral dendrite. This recording position allows mea-

surement of the true amplitude of the AD coupling without the

effect of attenuation by electrotonic axonal propagation (Fig-

ure 4C, bottom right). The coupling averaged 5.07 ± 0.31 mV

SEM (n = 24) but was subsequently corrected to 1.85 ±

0.11 mV SEM to take account for the amplification of the AD

coupling produced by a persistent sodium current (INa+P), which

is present in these afferents. (The correction was based on a

predicted amplification of 63.6% of the average AD coupling

amplitude from previous correlations of percent INa+P amplifica-

tion versus AD coupling amplitude at resting potential; see

Experimental Procedures; Curti and Pereda, 2004.)

We next considered the AD spike amplitude that is, on

average, most representative of that ‘‘seen’’ by the population

Figure 5. Voltage Dependence of Electrical Transmission

(A) The AD coupling is voltage dependent.

(B) Superimposed traces show the AD coupling potential recorded at resting potential (�73 mV) and near the threshold of the cell (–64 mV).

(C) Relationship between the antidromic coupling potential (AD coup, ordinates) and the presynaptic membrane potential (membrane potential, abscissa). The

dramatic increase in AD coupling with depolarization was blocked by QX-314 (Curti and Pereda, 2004), revealing a second voltage-dependent mechanism. See

also Figure S5.

(D) Only the QX-314-resistant mechanism is observed at the end of a 200 ms pulse.

(E) Superimposed traces show the AD coupling potential recorded at resting potential (–73 mV) near the threshold of the cell (–66 mV) and at �80 mV.

(F) Traces are illustrated scaled.

(G) Amplitude of AD coupling (obtained at the end of the 200ms pulse, red circles) and the afferent’s input resistance (blue boxes) versus membrane potential in a

representative experiment. Changes in amplitude of AD coup (K1) occur in the absence of changes in the afferent axon’s input resistance.

(H) Summary for ten experiments (red dots are AD coupling; blue squares are input resistance).

Neuron


of CEs. We reasoned that the amplitude of the AD spike at the

center of the terminal field of CEs in the lateral dendrite would

yield a good approximation. Because the amplitude of the M-

cell AD spike decays along the lateral dendrite (the M-cell spike

is generated at the axon initial segment and neither the soma nor

dendrite have active properties; Furshpan and Furukawa, 1962)

and because the precise location of the electrode in the dendrite

cannot be controlled, this AD spike amplitude varies between

experiments (10–20 mV). Therefore, to estimate the amplitude

of the AD spike at the center of the terminal field of CEs, where

most CEs terminate (Lin et al., 1983), we performed multiple

sequential recordings along the M-cell dendrite (Figure 4D).

Initial extracellular recordings were made in the M-cell axon

cap, which served as a ‘‘spatial calibration’’ marker, as the

distinctive field amplitude denotes the proximity of the electrode

to the initial segment (Furshpan and Furukawa, 1962). The elec-

trode was then moved at regularly spaced intervals along the

lateral dendrite for multiple recordings, during and after which

no changes were observed in the electrical properties of the

M-cell (Figure 4E). The amplitude of the AD spike decayed


Table 1. Values Used for Estimates of Gap Junctional Resistance

Presynaptic

Spike

Postsynaptic

Coupling)

Population Coupling

Coefficient

Postsynaptic Cell

Input Resistance

Junctional

Resistance

Orthodromic (CE to M-cell) 87.6 ± 0.9 mV,

n = 203

0.73 ± 0.04 mV,

n = 76

0.008 1.32 ± 0.3 MU, n = 9 168.3 MU

Antidromic (M-cell to CE) 10.6 mV 1.85 ± 0.11 mV,

n = 24

0.175 8.05 ± 0.74 MU, n = 20 39.8 MU

Mean ± SEM.

Neuron


exponentially (r2 = 0.99) with a space constant of�300 mm and a

predicted amplitude of 10.6 mV at the center of the terminal field

of CEs (which start�200 mm from the initial segment; Figure 4F).

These measurements yielded an antidromic CC of 0.175. The

input resistance of CEs was directly measured with current

pulses during intracellular recordings, with a resulting average

of 8.05 ± 0.74 MU SEM (n = 20).

Using these measurements and the equation described in the

Experimental Procedures, we obtained values of junctional

resistance of 168.3 MU in the orthodromic direction and of

39.8 MU in the antidromic direction (Table 1). This more than

4-fold difference between orthodromic and antidromic junctional

resistance indicates that electrical synapses at CEs rectify in a

way that enhances transmission of signals from the M-cell

dendrite into presynaptic afferents. While calculations were

based on values that we consider are the most accurate mea-

sures of the signals involved, the asymmetry in junctional resis-

tance was observed for a wide range of values, including the

average AD spike amplitude obtained during paired recordings

(which averaged 15.9 ± 0.48 mV SEM; n = 18) and presynaptic

spikes’ amplitudes recorded at the terminal (Figure S4), therefore

providing a high degree of confidence in the conclusion that GJs

between CEs and the M-cell rectify. In other words, electrical

rectification is sufficiently large to be detected by our indirect

experimental method. Accordingly, despite less favorable

experimental conditions for calculating accurate antidromic

CCs (and therefore for revealing GJ asymmetries), calculations

of GJ resistance obtained for each of the CEs illustrated in Fig-

ure S3, using the values of presynaptic spikes and coupling

potentials recorded at each of the afferents, still reveal an asym-

metry of GJ resistance (Figure S3C). Thus, the asymmetry of

electrical transmission observed between CEs and the M-cell

is supported by two contributing factors, an asymmetry of input

resistances between the coupled cells and an asymmetry of GJ

resistance (rectification).

Rectifying electrical synapses exhibit voltage-dependent

behavior (Furshpan and Potter, 1959; Giaume et al., 1987). We

have previously shown that the AD coupling potential produced

by the retrograde spread of the AD spike from the postsynaptic

M-cell is dramatically enhanced by depolarization of the presyn-

aptic terminal (Figure 5A; Pereda et al., 1995; Curti and Pereda,

2004). This dramatic voltage-dependent enhancement of electri-

cal coupling upon depolarization does not represent a property

of the junctions themselves but rather the activation of an

INa+P present at presynaptic terminals that acts to amplify the

synaptic response (Curti and Pereda, 2004). The interplay of

this current with an A-type repolarizing K+ conductance (IA)


generally reproduces the waveform of the coupling recorded at

resting potential (Figure 5B; Curti and Pereda, 2004), exhibits

an increased time to peak (Figure 5B), and the amplification is

blocked by both extracellular TTX and intracellular application

of QX-314 (changes occurred within a time window in which

the spikes of theCEs remained essentially unaffected; Figure 5C;

Curti and Pereda, 2004). Blockade of the INa+P reveals a

second, less prominent, voltage-dependent component that is

symmetrical relative to resting membrane potential. This second

voltage-dependent component can also be observed in the

absence of TTX and QX-314 at the end of a long (250 ms) depo-

larizing pulse (Figure 5D) when the above-mentioned conduc-

tances are no longer active, further indicating the existence of

two different voltage-dependent mechanisms (Curti and Pereda,

2004). Both components can also be isolated by curve fitting

(Figure S5). The QX-314-insensitive voltage-dependent behavior

had a slope of 0.094, equivalent to a change in AD coupling

amplitude of 3.81% per mV of membrane potential change,

which is symmetrical from resting potential, and unlike the

INa+P component, it does not modify the time to peak (Figure 5E)

nor the kinetics of the coupling potential (Figure 5F). We hypoth-

esized that the QX-314-insensitive voltage-dependent compo-

nent could correspond to either (1) a voltage-dependent

behavior of GJ channels or (2) a voltage-dependent behavior of

the cell’s membrane resistance, which could proportionally

modify the amplitude of the coupling potential. To distinguish

between these two possibilities, we measured both the ampli-

tude of the AD coupling potential and the CE’s input resistance

under different membrane potentials at the end of a 250 ms

pulse, where active conductances do not contribute to coupling

amplification. As illustrated in Figures 5G (single experiment) and

5H (n = 10), changes in amplitude of the AD coupling potential

were independent of the CE’s input resistance, which remained

constant through the full range of membrane potentials. As is the

case with other rectifying electrical synapses (Giaume and Korn,

1984), we found a difference between the resting potentials of

the coupled cells. The values averaged �71.7 ± 0.32 mV SEM

(n = 203) for CEs, where �74 mV was the most hyperpolarized

value, and �78.7 ± 2.5 mV SEM (n = 95; p < 0.01) for the

M-cell, where �85 mV was the most hyperpolarized value, sug-

gesting the existence of a transjunctional voltage of �10 mV, on

top of which electrical signals operate. Thus, we conclude that

electrical synapses at CEs exhibit voltage-dependence, where

depolarization of the presynaptic terminal enhances retrograde

electrical communication. By virtue of their electrical direction-

ality and voltage-dependence, heterotypic GJs in CEs act syner-

gistically with the presynaptic QX-314-sensitive component

Figure 6. Rectification Promotes Lateral ExcitationCartoon illustrates the lateral excitation of CE afferents on the M-cell lateral

dendrite and the contribution of electrical rectification to this phenomenon.

Dendritic synaptic potentials (yellow) evoked by suprathreshold electrical

stimulation of VIIIth nerve afferents (orange labeled CEs) spread to neighboring

subthreshold terminals (yellow labeled CEs). Electrical rectification favors the

retrograde transmission of dendritic signals toward CEs in a higher resistance

pathway (>Rn), counteracting the leak of currents toward the soma following a

pathway of low resistance (<Rn). Because of its voltage-dependent properties,

electrical transmission acts as a coincidence detector, facilitating the

recruitment of CEs that are already depolarized, such as during the invasion of

an incoming action potential, whose depolarization travels ahead several

nodes (bottom left CE; note that the arrow is bigger and the yellow in the CE

more intense, denoting the increase in coupling produced by presynaptic

depolarization).

Neuron


(INa+P) to promote cooperativity between afferents via lateral

excitation of neighboring terminals (Figure 6). This voltage-

dependence is also likely to affect anterograde transmission.

Heterotypic channels formed by expression of Cx35 and

Cx34.7 in oocytes exhibited voltage-dependent rectification of

instantaneous current (O’Brien et al., 1998), with properties

consistent with those observed at CE/M-cell contacts, although

the magnitude of the rectification was significantly smaller

(�30%). We re-examined the properties of Cx34.7/Cx35 junc-

tions by expressing these connexins in Rin cells (Figure S6A).

These heterotypic junctions showed both instantaneous and

steady-state properties (Figure S6D) similar to those reported

in oocytes (O’Brien et al., 1998). Such disparity between in vivo

and in vitro behaviors suggested, in addition to the molecular

asymmetry, a possible contribution of cell-specific factors to

generate rectification at either the CEs or M-cell, which could

include the association of ions and charged molecules with con-

nexin-specific residues in one of the hemichannels. We recently

reported that changes in free intracellular [Mg2+] modify the

properties of Cx36 GJ channels, both in cell expression systems

and native electrical synapses (Palacios-Prado et al., 2013). To

test the ability of Cx35 and Cx34.7 hemichannels to promote

rectification in the presence of [Mg2+], we asked if modifications

of free [Mg2+] in only one of the coupled cells (a reduction, in this

case, from 1 mM to 25 mM) could lead to asymmetry of electrical

coupling. This manipulation led to dramatic rectification of both

instantaneous and steady-state conductance-voltage relations

(Figures S6E and S6F). Both Cx34.7 and Cx35 sides were sensi-

tive to changes in free [Mg2+], but remarkably, theywere differen-

tially affected, both qualitatively and quantitatively (compare

instantaneous and steady-state responses in Figures S6E and

S6F). Although Mg2+ is unlikely to be the factor that enhances

rectification under physiological conditions at CE/M-cell synap-

ses, these findings demonstrate that (1) asymmetry of cytosolic

factors can induce rectification and that (2) molecular differences

in heterotypic junctions might contribute to a differential sensi-

tivity of each hemichannel to induce electrical rectification.

Thus, molecular asymmetry may be required but might not be

sufficient to generate strong rectification, and interactions with

cytosolic soluble factors could endow electrical synapses with

complex rectifying properties.

DISCUSSION

Heterotypic Channels Formed by Two Teleost Homologsof Cx36 Mediate Electrical Transmission at CEsWe have previously reported the presence of Cx35 at CEs and

suggested that intercellular channels were likely homotypic but

specifically noted the possible presence of other connexins at

these junctions (Pereda et al., 2003). We report here the

presence of Cx34.7, a second teleost homolog of Cx36 (O’Brien

et al., 1998), at CE/M-cell contacts. Our earlier results are never-

theless consistent with the detailed characterization of

antibodies we report here, which indicates that some of the

Cx36 antibodies previously used (i.e., Ab298) recognize both

Cx35 and Cx34.7, therefore labeling both pre- and postsynaptic

hemiplaques. Members of the connexin protein family can be

permissive or nonpermissive for forming functional intercellular

channels with each other. Heterotypic channels are especially

prominent among glial cells (Rash, 2010) and are found in various

tissues (Elenes et al., 2001), where they provide diversity for

intercellular communication (Rackauskas et al., 2007; Pala-

cios-Prado and Bukauskas, 2009). Heterotypic junctions at

CEs are somewhat unconventional, in that they are formed by

two teleost homologs of a connexin that is normally not permis-

sive for forming intercellular channels with any other connexins.

In tests of the capacity of Cx36 to form channels with ten other

connexin family members, Cx36 was permissive for channel for-

mation only with itself (Teubner et al., 2000). The limited amino

acid sequence difference between Cx34.7 and Cx35 appear

not to have caused sufficient structural changes to render these

connexins incompatible, and indeed, our data show that adult

CE/M-cell GJs gap junctions are formed exclusively from hetero-

typic coupling of these two connexins.

Heterotypic GJs between CEs and the M-cell RectifyAlthough the experimental access did not allow us to perform a

detailed biophysical analysis, our data indicate that these

rectifying junctions are associated with voltage-dependent

properties having kinetics similar to those at the classic crayfish

rectifying synapse (Furshpan and Potter, 1959; Giaume and

Korn, 1984). (These results contrast with a previous report sug-

gesting that electrical synapses at CEs do not rectify [Lin and

Faber, 1988]. The discrepancy with our estimates mainly arises

from differences in the values of AD coupling and dendritic input

resistance used for the calculations of junctional resistance that


Neuron


were critical for revealing the asymmetry.) Heterotypic channels

formed by recombinant Cx32 and Cx26 exhibit rectification

properties (Barrio et al., 1991; Rubin et al., 1992; Bukauskas

et al., 1995) that are reminiscent of those observed at rectifying

synapses in crayfish (Furshpan and Potter, 1959) and hatchetfish

(Auerbach and Bennett, 1969), indicating that molecular asym-

metry between hemichannels might constitute a principal

determinant of electrical rectification. Supporting Furshpan

and Potter’s hypothesis that junctional membranes behave as

a diode (electrical rectifier) rather than a simple electrical resistor

(Furshpan and Potter, 1959), biophysical modeling combined

with genetic analysis of heterotypic Cx32/Cx26 channels (Oh

et al., 1999) suggested that instantaneous rectification of electri-

cal coupling observed at these channels can be explained by the

separation of fixed positive (p) and negative (n) charges across

the junctional membrane, which results from the pairing of hemi-

channels with opposite charges at their channel, leading to the

formation of a diode or ‘‘p-n junction,’’ which was shown to be

capable of generating steep current-voltage relations (Oh

et al., 1999). We show here that, in addition to molecular asym-

metries, cytosolic-soluble cell-specific factors (such as Mg2+)

can contribute substantially to the generation of rectification in

electrical synapses (Figure S6). Furthermore, although both

Cx34.7 and Cx35 sides were sensitive to changes in [Mg2+],

they were differentially affected, indicating that molecular differ-

ences might contribute to a differential sensitivity of each hemi-

channel to soluble factors to enhance electrical rectification.

While Mg2+ is unlikely to be the factor creating rectification under

physiological conditions at CE/M-cell synapses, as yet undeter-

mined channel interacting cytosolic soluble factors (including

intracellular polyamines; Shore et al., 2001; Musa and Veenstra,

2003; Musa et al., 2004) may induce electrical rectification, either

because their concentrations are different on each side of the

junction (coupling in the M-cell occurs between two different

cell types and their intracellular milieus could be different) and/

or by preferentially interacting with hemichannels of one side of

the heterotypic junction. Finally, asymmetry could be also gener-

ated by differences in posttranslational modifications of the

apposing hemichannels, such as connexin phosphorylation,

which may contribute to rectifying properties by altering surface

charge or conformation of the proteins (Alev et al., 2008; O’Brien

et al., 1998).

Rectification Promotes Bidirectionality of ElectricalCommunicationAlthough closely associated with early evidence for electrical

transmission (Furshpan and Potter, 1959), electrical rectification

is an underestimated property of electrical synapses. Notably,

rectification is generally associated with unidirectionality of elec-

trical communication. Our results clearly separate the two

notions (rectification and directionality), as rectification in this

case acts to promote bidirectionality of electrical communica-

tion, which otherwise is challenged by the geometrical character-

istics and electrical properties of theM-cell andCEs.We suggest

that rectification, as in theM-cell, could alsounderlie bidirectional

communication between neuronal processes of dissimilar size

elsewhere, compensating for potentially challenging electrical

and geometrical conditions for the spread of currents.


The M-cell network mediates auditory-evoked tail-flip

escape responses in teleost fish, and much data support CEs

as having a primary role in generating these responses (Faber

and Pereda, 2011). Because electrical synapses at CEs are

bidirectional, signals originating in the M-cell dendrite can influ-

ence CE excitability (Pereda et al., 1995). We propose that

retrograde transmission is relevant functionally based on the

following: (1) it allows CEs to be electrically coupled to each

other through the lateral dendrite of the M-cell (Figure 6; Per-

eda et al., 1995); (2) this coupling serves as a mechanism for

‘‘lateral excitation’’ (Pereda et al., 1995; Herberholz et al.,

2002; DeVries et al., 2002); (3) lateral excitation promotes the

coordinated activity of a population of CEs; and (4) the coordi-

nated activity likely increases efficacy of auditory input for the

initiation of an escape response. These events are likely

enhanced by electrical rectification, which favors the spread

of currents from the M-cell lateral dendrite toward the presyn-

aptic CEs. That is, because dendritic currents would encounter

a lower resistance to spread across these junctions than those

generated presynaptically, electrical rectification favors the

retrograde transmission of dendritic signals, counteracting the

leak of currents toward the soma, following a pathway of low

resistance (Figure 6). Moreover, given that coupling increases

with presynaptic depolarization, the voltage dependence of

electrical coupling we describe here acts as a ‘‘coincidence

detector,’’ promoting the recruitment of CEs that are already

depolarized, such as during the invasion of an incoming action

potential, whose depolarization (because of cable properties)

travels several nodes ahead without reaching threshold (Fig-

ure 6). Thus, although differences in input resistance signifi-

cantly contribute to the asymmetry of electrical transmission

between these cells, rectification plays a critical functional

role by directing currents toward the presynaptic endings.

Are Hemiplaques in Electrical Synapses the MirrorImages of Each Other?A generalized perception is that each side in most GJ plaques

represents themirror image of the other, as its formation requires

the symmetric arrangement of hemichannels. This view, how-

ever, is changing with the recognition that connexins associate

with a variety of proteins, resulting in the formation of macromo-

lecular complexes (Herve et al., 2012). Furthermore, electrical

synapses have been shown to be dynamic structures, where

connexins actively turnover (Flores et al., 2012) and exhibit activ-

ity-dependent regulation of their coupling strength (Yang et al.,

1990; Pereda and Faber, 1996; Landisman and Connors, 2005;

Cachope et al., 2007; Haas et al., 2011). These properties

suggest that each side in a GJ plaque must be supported by a

scaffold structure, similar to postsynaptic densities at chemical

synapses (Kennedy, 2000). While the detailed composition of

this scaffold is largely unknown, several molecules interact

with Cx36 (Li et al., 2004, 2009; Burr et al., 2005; Ciolofan

et al., 2006; Alev et al., 2008) and its teleost homologs (Flores

et al., 2008, 2010). Thus, molecular diversity in electrical synap-

ses might not only be endowed by the connexins present but

also potentially by differences in the ensemble of scaffold and

regulatory molecules associated with each side of the gap junc-

tions that form these synapses, which could be an additional

Neuron


means of creating molecular asymmetry, impacting on the func-

tional properties of these channels.

EXPERIMENTAL PROCEDURES

For full methodological details see the Supplemental Experimental

Procedures.

Immunohistochemistry

Goldfish brains were fixed and sections were incubated simultaneously with

rabbit polyclonal anti-Cx34.7 IL antibody (see Table S1) and mouse

monoclonal anti-Cx35 (Chemicon MAB3043) antibody, incubated with Alexa

Fluor 488-conjugated goat anti-rabbit and/or Alexa Fluor 594-conjugated

goat anti-mouse secondary antibodies, and coverslipped using n-propyl

gallate-based mounting media.

Confocal Microscopy and Image Processing

Sections were imaged using anOlympusBX61WI confocal microscope. Image

analysis was performed using Image J (National Institutes of Health [NIH]) and

MetaMorph software. Colocalization of Cx35 andCx34.7wasmeasured as the

percentage of the area labeled for Cx35 that was also labeled for Cx34.7 and

the converse.

Antibodies

The antibodies used are listed in Table S1 along with their species of origin,

designation, epitope recognition, source, and characteristics of detection of

either or both Cx34.7 and Cx35.

Freeze-Fracture Replica Immunogold Labeling

Specimens were processed by single-replica FRIL and one additional spec-

imen by double-replica SDS-FRIL (sodium dodecyl sulfate-digested fracture

replica labeling, which we designate as DR-FRIL). For single-replica samples,

a gold ‘‘index’’ grid (aka ‘‘Finder’’ grid) was bonded to the coated surfaces

using Lexan plastic (polycarbonate plastic) dissolved in dichloroethane; the

samples were thawed and ‘‘grid-mapped’’ by confocal microscopy, with

which the location of the M-cell lateral dendrite was determined.

Double-Replica FRIL

A 150-mm-thick slice of goldfish hindbrain containing a Lucifer Yellow-injected

M-cell was cryoprotected and fractured at �105�C in a prototype JFD-2

freeze-etch machine equipped with a turbopump but lacking a liquid-nitro-

gen-cooled shroud. The opened double-replica ‘‘sandwich’’ was coated with

3–5 nm of carbon, 1.5 nm of platinum, and �20 nm of carbon.

Electrophysiology

Surgical and recording techniques were similar to those described previously

(Smith and Pereda, 2003; Curti and Pereda, 2004). Intracellular recordings

were obtained in vivo from the lateral dendrite; both current clamp and sin-

gle-electrode voltage clamp techniques were employed. Individual VIIIth nerve

afferents were penetrated, either at the posterior VIIIth root during simulta-

neous recordings with the M-cell’s lateral dendrite or less often, intracranially,

close to the dendrite.

Estimate of Junctional Resistance

Junctional resistance in each direction was estimated following Devor and

Yarom (2002). These estimates of the junctional resistance assume a simple

two neuron model with passive membrane properties coupled directly by a

single junction.

In Vitro Electrophysiological Measurements

Experiments were performed on Rin cells expressing Cx35 or Cx34.7

tagged with eYFP or DsRed, respectively. To adjust the concentration of

intracellular free Mg2+, we used pipette solutions containing different concen-

trations of MgCl2 and EDTA and the web-based Maxchelator software

(http://www.stanford.edu/�cpatton/webmaxcS.htm) to calculate free ionic

concentrations.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

six figures, and one table and can be found with this article online at http://

dx.doi.org/10.1016/j.neuron.2013.06.037.

ACKNOWLEDGMENTS

We thank Michael V.L. Bennett and Peter Sterling for their comments on

the manuscript. This research was supported by National Institutes of

Health grants S10RR05831, S10RR08329, NS044395, and NS044010 (to

J.E.R.), EY012857 (to J.O.), RO1NS072238 (to F.F.B.), Canadian Institute for

Health Research (to J.I.N.), and by NIH grants DC03186, DC011099,

R21NS055726, and NS0552827 (to A.E.P.).

Accepted: June 13, 2013

Published: September 4, 2013

REFERENCES

Alev, C., Urschel, S., Sonntag, S., Zoidl, G., Fort, A.G., Hoher, T., Matsubara,

M., Willecke, K., Spray, D.C., and Dermietzel, R. (2008). The neuronal con-

nexin36 interacts with and is phosphorylated by CaMKII in a way similar to

CaMKII interaction with glutamate receptors. Proc. Natl. Acad. Sci. USA

105, 20964–20969.

Auerbach, A.A., and Bennett, M.V. (1969). A rectifying electrotonic synapse in

the central nervous system of a vertebrate. J. Gen. Physiol. 53, 211–237.

Barrio, L.C., Suchyna, T., Bargiello, T., Xu, L.X., Roginski, R.S., Bennett, M.V.,

and Nicholson, B.J. (1991). Gap junctions formed by connexins 26 and 32

alone and in combination are differently affected by applied voltage. Proc.

Natl. Acad. Sci. USA 88, 8410–8414.

Bennett, M.V. (1966). Physiology of electrotonic junctions. Ann. N Y Acad. Sci.

137, 509–539.

Bennett, M.V.L. (1977). Electrical transmission, a functional analysis and com-

parison with chemical transmission. In Cellular Biology of Neurons, Volume I,

The Handbook of Physiology - The Nervous System I, E.R. Kandel, ed.

(Baltimore: Williams and Wilkins), pp. 357–416.

Bukauskas, F.F., Elfgang, C., Willecke, K., and Weingart, R. (1995).

Heterotypic gap junction channels (connexin26-connexin32) violate the para-

digm of unitary conductance. Pflugers Arch. 429, 870–872.

Burr, G.S., Mitchell, C.K., Keflemariam, Y.J., Heidelberger, R., and O’Brien, J.

(2005). Calcium-dependent binding of calmodulin to neuronal gap junction

proteins. Biochem. Biophys. Res. Commun. 335, 1191–1198.

Cachope, R., Mackie, K., Triller, A., O’Brien, J., and Pereda, A.E. (2007).

Potentiation of electrical and chemical synaptic transmission mediated by

endocannabinoids. Neuron 56, 1034–1047.

Ciolofan, C., Li, X.B., Olson, C., Kamasawa, N., Gebhardt, B.R., Yasumura, T.,

Morita, M., Rash, J.E., and Nagy, J.I. (2006). Association of connexin36 and

zonula occludens-1 with zonula occludens-2 and the transcription factor

zonula occludens-1-associated nucleic acid-binding protein at neuronal gap

junctions in rodent retina. Neuroscience 140, 433–451.

Condorelli, D.F., Parenti, R., Spinella, F., Trovato Salinaro, A., Belluardo, N.,

Cardile, V., and Cicirata, F. (1998). Cloning of a new gap junction gene

(Cx36) highly expressed in mammalian brain neurons. Eur. J. Neurosci. 10,

1202–1208.

Connors, B.W., and Long, M.A. (2004). Electrical synapses in the mammalian

brain. Annu. Rev. Neurosci. 27, 393–418.

Cruciani, V., and Mikalsen, S.O. (2007). Evolutionary selection pressure and

family relationships among connexin genes. Biol. Chem. 388, 253–264.

Curti, S., and Pereda, A.E. (2004). Voltage-dependent enhancement of electri-

cal coupling by a subthreshold sodium current. J. Neurosci. 24, 3999–4010.

Curti, S., Gomez, L., Budelli, R., and Pereda, A.E. (2008). Subthreshold sodium

current underlies essential functional specializations at primary auditory affer-

ents. J. Neurophysiol. 99, 1683–1699.


http://www.stanford.edu/%7Ecpatton/webmaxcS.htm

http://www.stanford.edu/%7Ecpatton/webmaxcS.htm



Neuron


Devor, A., and Yarom, Y. (2002). Electrotonic coupling in the inferior olivary

nucleus revealed by simultaneous double patch recordings. J. Neurophysiol.

87, 3048–3058.

DeVries, S.H., Qi, X., Smith, R., Makous, W., and Sterling, P. (2002). Electrical

coupling between mammalian cones. Curr. Biol. 12, 1900–1907.

Eastman, S.D., Chen, T.H., Falk, M.M., Mendelson, T.C., and Iovine, M.K.

(2006). Phylogenetic analysis of three complete gap junction gene families re-

veals lineage-specific duplications and highly supported gene classes.

Genomics 87, 265–274.

Elenes, S., Martinez, A.D., Delmar, M., Beyer, E.C., and Moreno, A.P. (2001).

Heterotypic docking of Cx43 and Cx45 connexons blocks fast voltage gating

of Cx43. Biophys. J. 81, 1406–1418.

Faber, D.S., and Pereda, A.E. (2011). Physiology of the Mauthner Cell,

Functions. In Encyclopedia of Fish Physiology, From Genome to

Environment, A.P. Farrell, ed. (San Diego: Academic Press), pp. 73–79.

Flores, C.E., Li, X., Bennett, M.V., Nagy, J.I., and Pereda, A.E. (2008).

Interaction between connexin35 and zonula occludens-1 and its potential

role in the regulation of electrical synapses. Proc. Natl. Acad. Sci. USA 105,

12545–12550.

Flores, C.E., Cachope, R., Nannapaneni, S., Ene, S., Nairn, A.C., and Pereda,

A.E. (2010). Variability of distribution of Ca(2+)/calmodulin-dependent kinase II

at mixed synapses on the mauthner cell: colocalization and association with

connexin 35. J. Neurosci. 30, 9488–9499.

Flores, C.E., Nannapaneni, S., Davidson, K.G., Yasumura, T., Bennett, M.V.,

Rash, J.E., and Pereda, A.E. (2012). Trafficking of gap junction channels at a

vertebrate electrical synapse in vivo. Proc. Natl. Acad. Sci. USA 109, E573–

E582.

Fujimoto, K. (1995). Freeze-fracture replica electron microscopy combined

with SDS digestion for cytochemical labeling of integral membrane proteins.

Application to the immunogold labeling of intercellular junctional complexes.

J. Cell Sci. 108, 3443–3449.

Fukami, Y., Furukawa, T., and Asada, Y. (1965). Excitability changes of the

Mauthner cell during collateral inhibition. J. Gen. Physiol. 48, 581–600.

Furshpan, E.J., and Potter, D.D. (1959). Transmission at the giant motor syn-

apses of the crayfish. J. Physiol. 145, 289–325.

Furshpan, E.J., and Furukawa, T. (1962). Intracellular and extracellular re-

sponses of the several regions of the Mauthner cell of the goldfish.

J. Neurophysiol. 25, 732–771.

Giaume, C., and Korn, H. (1984). Voltage-dependent dye coupling at a recti-

fying electrotonic synapse of the crayfish. J. Physiol. 356, 151–167.

Giaume, C., Kado, R.T., and Korn, H. (1987). Voltage-clamp analysis of a cray-

fish rectifying synapse. J Physiol. 386, 91–112.

Haas, J.S., Zavala, B., and Landisman, C.E. (2011). Activity-dependent long-

term depression of electrical synapses. Science 334, 389–393.

Herberholz, J., Antonsen, B.L., and Edwards, D.H. (2002). A lateral excitatory

network in the escape circuit of crayfish. J. Neurosci. 22, 9078–9085.

Herve, J.C., Derangeon,M., Sarrouilhe, D., Giepmans, B.N., and Bourmeyster,

N. (2012). Gap junctional channels are parts of multiprotein complexes.

Biochim. Biophys. Acta 1818, 1844–1865.

Kennedy, M.B. (2000). Signal-processing machines at the postsynaptic den-

sity. Science 290, 750–754.

Landisman, C.E., and Connors, B.W. (2005). Long-term modulation of electri-

cal synapses in the mammalian thalamus. Science 310, 1809–1813.

Li, X., Olson, C., Lu, S., Kamasawa, N., Yasumura, T., Rash, J.E., and Nagy, J.I.

(2004). Neuronal connexin36 association with zonula occludens-1 protein (ZO-

1) in mouse brain and interaction with the first PDZ domain of ZO-1. Eur. J.

Neurosci. 19, 2132–2146.

Li, X., Lu, S., and Nagy, J.I. (2009). Direct association of connexin36 with zon-

ula occludens-2 and zonula occludens-3. Neurochem. Int. 54, 393–402.

Lin, J.W., and Faber, D.S. (1988). Synaptic transmission mediated by single

club endings on the goldfish Mauthner cell. I. Characteristics of electrotonic

and chemical postsynaptic potentials. J. Neurosci. 8, 1302–1312.


Lin, J.W., Faber, D.S., and Wood, M.R. (1983). Organized projection of the

goldfish saccular nerve onto the Mauthner cell lateral dendrite. Brain Res.

274, 319–324.

Musa, H., and Veenstra, R.D. (2003). Voltage-dependent blockade of con-

nexin40 gap junctions by spermine. Biophys. J. 84, 205–219.

Musa, H., Fenn, E., Crye, M., Gemel, J., Beyer, E.C., and Veenstra, R.D. (2004).

Amino terminal glutamate residues confer spermine sensitivity and affect

voltage gating and channel conductance of rat connexin40 gap junctions.

J. Physiol. 557, 863–878.

O’Brien, J., Bruzzone, R., White, T.W., Al-Ubaidi, M.R., and Ripps, H. (1998).

Cloning and expression of two related connexins from the perch retina define

a distinct subgroup of the connexin family. J. Neurosci. 18, 7625–7637.

Oh, S., Rubin, J.B., Bennett, M.V., Verselis, V.K., and Bargiello, T.A. (1999).

Molecular determinants of electrical rectification of single channel conduc-

tance in gap junctions formed by connexins 26 and 32. J. Gen. Physiol. 114,

339–364.

Orthmann-Murphy, J.L., Freidin, M., Fischer, E., Scherer, S.S., and Abrams,

C.K. (2007). Two distinct heterotypic channels mediate gap junction coupling

between astrocyte and oligodendrocyte connexins. J. Neurosci. 27, 13949–

13957.

Palacios-Prado, N., and Bukauskas, F.F. (2009). Heterotypic gap junction

channels as voltage-sensitive valves for intercellular signaling. Proc. Natl.

Acad. Sci. USA 106, 14855–14860.

Palacios-Prado, N., Hoge, G., Marandykina, A., Rimkute, L., Chapuis, S.,

Paulauskas, N., Skeberdis, V.A., O’Brien, J., Pereda, A.E., Bennett, M.V.,

and Bukauskas, F.F. (2013). Intracellular magnesium-dependent modulation

of gap junction channels formed by neuronal connexin36. J. Neurosci. 33,

4741–4753.

Pereda, A.E., and Faber, D.S. (1996). Activity-dependent short-term enhance-

ment of intercellular coupling. J. Neurosci. 16, 983–992.

Pereda, A.E., Bell, T.D., and Faber, D.S. (1995). Retrograde synaptic commu-

nication via gap junctions coupling auditory afferents to the Mauthner cell.

J. Neurosci. 15, 5943–5955.

Pereda, A., O’Brien, J., Nagy, J.I., Bukauskas, F., Davidson, K.G., Kamasawa,

N., Yasumura, T., and Rash, J.E. (2003). Connexin35mediates electrical trans-

mission at mixed synapses on Mauthner cells. J. Neurosci. 23, 7489–7503.

Pereda, A.E., Rash, J.E., Nagy, J.I., and Bennett, M.V. (2004). Dynamics of

electrical transmission at club endings on the Mauthner cells. Brain Res.

Brain Res. Rev. 47, 227–244.

Phelan, P., Goulding, L.A., Tam, J.L., Allen, M.J., Dawber, R.J., Davies, J.A.,

andBacon, J.P. (2008). Molecularmechanism of rectification at identified elec-

trical synapses in the Drosophila giant fiber system. Curr. Biol. 18, 1955–1960.

Rackauskas, M., Verselis, V.K., and Bukauskas, F.F. (2007). Permeability of

homotypic and heterotypic gap junction channels formed of cardiac connexins

mCx30.2, Cx40, Cx43, and Cx45. Am. J. Physiol. Heart Circ. Physiol. 293,

H1729–H1736.

Rash, J.E. (2010). Molecular disruptions of the panglial syncytium block potas-

sium siphoning and axonal saltatory conduction, pertinence to neuromyelitis

optica and other demyelinating diseases of the central nervous system.

Neuroscience 168, 982–1008.

Rubin, J.B., Verselis, V.K., Bennett, M.V., and Bargiello, T.A. (1992). Molecular

analysis of voltage dependence of heterotypic gap junctions formed by con-

nexins 26 and 32. Biophys. J. 62, 183–193.

Shore, L., McLean, P., Gilmour, S.K., Hodgins, M.B., and Finbow, M.E. (2001).

Polyamines regulate gap junction communication in connexin 43-expressing

cells. Biochem. J. 357, 489–495.

Smith, M., and Pereda, A.E. (2003). Chemical synaptic activity modulates

nearby electrical synapses. Proc. Natl. Acad. Sci. USA 100, 4849–4854.

Teubner, B., Degen, J., Sohl, G., Guldenagel, M., Bukauskas, F.F., Trexler,

E.B., Verselis, V.K., De Zeeuw, C.I., Lee, C.G., Kozak, C.A., et al. (2000).

Functional expression of the murine connexin 36 gene coding for a neuron-

specific gap junctional protein. J. Membr. Biol. 176, 249–262.

Neuron


Tuttle, R., Masuko, S., and Nakajima, Y. (1986). Freeze-fracture study of the

large myelinated club ending synapse on the goldfish Mauthner cell: special

reference to the quantitative analysis of gap junctions. J. Comp. Neurol. 246,

202–211.

Volff, J.N. (2005). Genome evolution and biodiversity in teleost fish. Heredity

(Edinb) 94, 280–294.

Yang, X.D., Korn, H., and Faber, D.S. (1990). Long-term potentiation of electro-

tonic coupling at mixed synapses. Nature 348, 542–545.

Yum, S.W., Zhang, J., Valiunas, V., Kanaporis, G., Brink, P.R., White, T.W., and

Scherer, S.S. (2007). Human connexin26 and connexin30 form functional het-

eromeric and heterotypic channels. Am. J. Physiol. Cell Physiol. 293, C1032–

C1048.


molecular and functional asymmetry at a vertebrate ...€¦ · molecular and functional asymmetry...

Documents