Gap junction-mediated electrical transmission: regulatory mechanisms and plasticity

Abstract

The term synapse applies to cellular specializations that articulate the processing of information within neural circuits by providing a mechanism for the transfer of information between two different neurons. There are two main modalities of synaptic transmission: chemical and electrical. While most efforts have been dedicated to the understanding of the properties and modifiability of chemical transmission, less is still known regarding the plastic properties of electrical synapses, whose structural correlate is the gap junction. A wealth of data indicates that, rather than passive intercellular channels, electrical synapses are more dynamic and modifiable than was generally perceived. This article will discuss the factors determining the strength of electrical transmission and review current evidence demonstrating its dynamic properties. Like their chemical counterparts, electrical synapses can also be plastic and modifiable. This article is part of a Special Issue entitled: The Communicating junctions, roles and dysfunctions.

FIGURE 1. Neuronal gap junctions constitute the basis for electrical synaptic transmission

A, Early electron micrograph of a Club ending in goldfish revealing zones of close membrane apposition (arrows, inset), or “synaptic discs”, later shown to be gap junction plaques. Calibration is 1 μm. Modified from Robertson et al. (1963) [2]. B, High magnification of junctions between dendrites of spinal electromotor neurons in mormyrid fish. The plane of section is nearly perpendicular to the membranes and the overall thickness is about 140 A. Calibrations are 200 A. Adapted from Bennett et al. (1967) [151].

FIGURE 2. Factors that determine the strength of electrical transmission

A, The cartoon represents a pair of coupled neurons on which an equivalent circuit of the key elements that determines strength of coupling was superimposed. The junctional conductance (Gap Junctional Conductance) is represented by a lumped resistance (Rc), whereas the passive properties of coupled cells are represented by a resistor (R1, R2) in parallel with a capacitor, which together determine the input resistance and the time constant of the neuron. B, The steady state coupling coefficient. The strength of electrical synapses can be assessed by the injection of long polarizing current pulses in one of a pair of coupled neurons. Current injection evokes a voltage drop in the presynaptic cell (Pre), which is typically accompanied by a change of the membrane potential in the coupled cell of lower amplitude, slower temporal course and similar polarity. The duration of the current pulses is usually long enough to overcome the initial attenuation of the membrane potential produced by the filtering properties imposed by the passive properties of the postsynaptic membrane, so voltage changes can be measured at “steady state”. The strength of coupling is quantified by calculating the Coupling Coefficient (CC), defined as the ratio between the voltage deflections in the post- and presynaptic cells. As it can be observed, at steady state conditions this coefficient critically depends on the resistance of the postsynaptic cell. C, Constraints imposed by the time constant of the postsynaptic cell. Due to low-pass filtering properties of the coupled neurons, time varying signals are attenuated according to their frequency content. Left, Injection of sine wave current in the presynaptic cell (I_Pre) evokes a sinusoidal variation of the membrane potential of the presynaptic cell (V_Pre) that can be recorded as a coupling potential in the postsynaptic cell (V_Post). Right, Frequency transfer characteristics determined for pairs of electrically coupled fast-spiking (FS) and low-threshold spiking (LTS) inhibitory interneurons of neocortical layer 4 in the rat. Magnitude of transfer represents the ratio of membrane potential amplitude in the postsynaptic cell over that of the presynaptic cell, during sine wave injections of various frequencies at the presynaptic side, indicates that electrical contacts between pairs of FS and LTS interneurons behaves as low-pass filters. These results indicate that the strength of electrical coupling is stronger for presynaptic signals of lower frequency content. Modified from Gibson et al. (2005) [152].

FIGURE 3. Regulation of the electrical coupling y changes of the non-junctional membrane

A, The Inferior Olive glomerulus: Inhibitory (GABA, blue) and excitatory (Glutamate, yellow) synapses terminate in the vicinity of gap-junctions between dendrodendritic processes (spine) of inferior olivary neurons (pink). Inhibitory synaptic conductances are thought to briefly shunt excitatory currents, temporarily reducing effective electrotonic coupling between IO neurons. B – D, Optical recordings with high speed voltage-sensitive dye imaging, in vitro, describe patterns of ensemble oscillatory activity in the Inferior Olive. B, Optical recordings were cut into fragments of two or three oscillatory cycles and averaged based on the temporal profile of intracellular recordings (see Ref. for details). C, Application of the GABA_A receptor blocker picrotoxin enhances oscillatory clusters. D, These clusters are absent in slices treated with the gap-junction blocker 18β-glycyrrhetinic acid (18β-GA). The three representative images show the location and spatial spread of 1 oscillatory cycle. The amplitude and time-course of the optical responses for 1 pixel are also shown (asterisk in each left image). The time points for each image are labeled on the pixel trace; the start of the oscillatory cycle was defined as 0 ms. Modified from Leznik and Llinas (2005) [69].

FIGURE 4. Enhancement of electrical coupling by intrinsic membrane properties

A, Auditory afferents (Club Endings) terminate as mixed synapses (Mixed synapse) on the lateral dendrite of the goldfish Mauthner cell. The action potential produced by the antidromic stimulation of the Mauthner cell axon in the spinal cord (M-cell AD spike) can be recorded as a coupling potential in the presynaptic afferents (AD coupling). B, The amplitude of the AD coupling is voltage-dependent; it increases with presynaptic membrane depolarization and decreases with membrane hyperpolarization (current pulses of ± 0.9 nA). C, Superimposed traces illustrate the AD potential obtained at resting potential (RP, −72 mV) and at a depolarized potential (−65 mV). Enhancement of AD coupling results from the interplay between a subthreshold Na+ current (red), that adds to the depolarization produced by the coupling, and a delayed repolarizing K⁺ conductance (blue), which by terminating the action of the subthreshold Na⁺ current, reproduces the waverform of the AD coupling obtained at resting potential. Modified from Curti and Pereda (2004) [31]. D, Voltage-dependent amplification of coupling between pairs of electrically coupled Golgi cells in the rat cerebellar cortex. Depolarizing coupling potentials recorded in the postsynaptic cell (Post) in response to action potentials generated in the presynaptic cell (Pre). Recordings were obtained in control conditions (left), and when the Na⁺ channel blocker QX-314 was included in the recording electrode of the postsynaptic cell (right). In control conditions, depolarizing coupling potentials were progressively bigger with postsynaptic cell depolarization. Such amplification was absent when QX-314 was added to the postsynaptic recording electrode, suggesting the participation of a subthreshold Na⁺ current whose activation at subthreshold membrane potentials enhances coupling potential amplitude. Because of the pronounced afterhyperpolarization of the presynaptic action potential, electrical coupling was predominantly hyperpolarizing at hyperpolarized membrane potentials, whereas it became depolarizing as a result of the action of the subthreshold Na⁺ current, at more depolarized potentials. This sign reversal was blocked by QX-314, indicating the critical functional role of the subthreshold Na⁺ current (Fig. 4E). Modified from Dugue et al. (2009) [72].

FIGURE 5

A, Intrinsic membrane properties enhance the transfer of relatively high-frequency signals IR-DIC image of a pair of contiguous MesV neurons during a simultaneous whole-cell recording. B, Frequency transfer properties in a pair of electrically coupled MesV neurons using a sinusoidal current of increasing frequency (zap). Magnitude of the transfer under control conditions and after the extracellular application of TTX and 4-AP. The amplitude of the frequency-transfer characteristics was calculated as the ratio of the Fast Fourier Transform (FFT) of the postsynaptic cell over the FFT of the presynaptic cell. The solid pink area represents the difference in transfer in the two conditions, illustrating the contribution of active mechanisms to frequency-transfer characteristics, particularly at ~50 Hz (arrowhead). Modified from Curti el al., 2012 [74].

FIGURE 6. Neuromodulators regulate electrical transmission

A, Dopamine reduces junctional conductance between pairs of catfish horizontal cells in a dose-dependent fashion. Dopamine (1, 10 and 100 nM) was added to the extracellular solution during the intervals indicated by the red bars. Junctional conductance (nS) is plotted as a function of time. Modified from DeVries and Schwartz (1988) [80]. B, Electrical coupling between stratum lacunosum-moleculare interneurons is modulated by α-adrenergic receptors. Application of noradrenaline (20 μM) reduces the junctional conductance and the coupling coefficient. C, Time course of the effects of noradrenaline on the normalized junctional conductance and the coupling coefficient and input resistance. Modified from Zsiros and Maccaferri (2008) [95].

FIGURE 7. Activity-dependent plasticity of electrical transmission a primary auditory afferents on the goldfish Mauthner cell

A, Club endings exhibit mixed synaptic transmission. Typical experimental arrangement showing VIIIth nerve auditory primary afferents (which contact saccular hair cells; “hair cell”) terminating as Club endings on the ipsilateral Mauthner cell lateral dendrite. VIIIth nerve and postsynaptic electrodes are indicated. Inset: cartoon represents a Club ending, at which both mechanisms of synaptic transmission, electrical (gap junction) and chemical, coexist. VIIIth nerve stimulation evokes mixed (electrical and chemical) EPSPs (red trace). Here and elsewhere, unless otherwise indicated, each trace represents the average of at least 20 individual responses. B, Discontinuous tetanic stimulation (trains of six pulses at 500 Hz, every 2 s for 4 min; “500 Hz protocol”) of the VIIIth nerve can evoke persistent potentiation of both components of the EPSP. Plots here and in subsequent panels illustrate the amplitudes of the electrical and chemical components vs. time (each point represents the average of 20 traces) for a single experiment. C, Schematic representation of the proposed potentiating pathway. Ca⁺⁺ entering through NMDA receptors activates CaMKII that phosphorylates either glutamate receptors and connexins or regulatory molecules. Modified from Pereda et al. (1998) [110]. D, Repetitive stimulation of the posterior VIIIth nerve (100 Hz during 1 sec.; “100 Hz protocol”) evoked robust potentiation of both components of the mixed EPSP (n = 5). E, Model for endocannabinoid-mediated potentiation of electrical and chemical synaptic transmission at Club endings. Synaptic activity leads to mGluR activation paired with postsynaptic membrane depolarization, triggering endocannabinoid (eCB) release from the postsynaptic Mauthner cell dendrite, which activates CB1Rs on dopaminergic fibers. CB1R activation leads to dopamine release that, by activating postsynaptic D1/5 receptors, increases PKA activity responsible for simultaneous potentiation of electrical and glutamatergic (GluR) synaptic transmission. Modified from Cachope et al. (2007) [122].

FIGURE 8. Neuronal gap-junctions are in close proximity to glutamatergic synapses and show variability in coupling strength

A, Freeze-fracture immunogold labeling (FRIL) from goldfish Club endings shows Cx35 (10-nm gold beads) in a gap-junction plaque (pink). A nearby aggregate of E-face particles (yellow) represents a post-synaptic density for a glutamatergic synapse and shows labeling for the NR1 subunit (18-nm gold bead), indicating the presence of an NMDA receptor. B, Unitary EPSPs measured in the same Mauthner cell dendrite, but evoked from different club endings, show variability in the electrical conductance of the synaptic transmission, even though the pre-synaptic action potentials were highly invariable (not shown). Only one of the synaptic potentials shows a clear chemical component. Thus, electrical synapses from neighboring Club endings coexist at different degrees of conductance. C, Tracer coupling between the Mauthner cell (M-cell) and neighboring Club endings shows a similar diversity of coupling strength. The image shows that Neurobiotin injected to the Mauthner cell transferred to neighboring club endings (arrowheads) with different degrees of staining intensity, indicating that the junctions differ in their permeability. Thus, Club ending synapses on the goldfish Mauthner cell coexist and different degrees of conductance (panel B) and permeability (panel C), likely because of the regulation from nearby glutamatergic synapses (panel A, Fig. 6). A similar arrangement and coexistence of variable gap-junction strengths occurs in mammals, suggesting mechanisms similar to those in goldfish may function to modulate junctional conductance in mammals. Modified from Pereda et al. (2004) [105]. D, FRIL double labeling of Cx36 and NR1 in a rat inferior olivary neuron. The image shows a PSD (yellow) of a glutamatergic synapse with labeling for the NR1 subunit of an NMDA receptor (10nm-gold bead). The gap-junction plaque (pink) shows labeling for Cx36 (20nm-gold bead). This close arrangement of a Cx36-containing gap junction and an NR1-containing PSD is very similar to the close arrangement found in goldfish club endings (panel A). E, In the rat inferior olive, the labeling intensity of the somata of coupled cells (arrowheads) is highly variable. The image corresponds to a confocal projection (average of 17 z-sections) illustrating a neurobiotin-injected Inferior Olive neuron with multiple indirectly labeled neurons. Darker silhouettes represent more intense neurobiotin labeling; the variable labeling in the inferior olive is similar to the variable labeling of goldfish Club endings (panel C). Modified from Hoge et al. (2010) [123].

FIGURE 9. Effects of high-frequency stimulation on gap junctional strength at thalamic neurons

A, IR-DIC image of a thalamocortical slice with recording arrangement. Inset: High-magnification view of recorded neurons in TRN. Scale bars indicate 1 mm and 20 μm, respectively. B, Voltage responses to hyperpolarizing current pulses in the presynaptic cell (TRN 1) and coupling responses in cell 2 (TRN 2) before and after high-frequency stimulation of cortico-thalamic glutamatergic afferents. C, Time-course of coupling coefficients (black squares) and estimated junctional conductance (gray open circles) for nine pairs of cells before and after high-frequency stimulation. The induction of long-term depression was prevented by superfusing with MCPG, an antagonist of mGLUR receptors. Modified from Landisman and Connors, 2003 [103].

FIGURE 10. Connexin-associated proteins in electrical synapses

A, Cx35 and ZO-1 co-localize at Club Endings. Laser scanning confocal projection of an individual Club ending after double immunolabeling showing intense punctate labeling for Cx35 and ZO-1 (right panels) and high levels of co-localization in yellow (left panel). C, In contrast, αCaMKII labeling is more diffuse and highly variable between contiguous Club endings. While αCaMKII labeling was always observed at the periphery, where PSDs are located, it is also found at the center of the Club endings contact area in some cases (compare top Club endings with the bottom one). D, The extensive co-localization of Cx35 with ZO-1 suggests that this scaffold protein could constitute a structural component of gap junctions at these terminals. Activity of neighboring chemically transmitting regions within the terminal trigger changes in junctional conductance, via a PSD-mediated mechanism (arrows) promoting the association of CaMKII to Cx35 and ZO-1. The association of CaMKII to electrical synapses would be thus non-obligatory and driven by synaptic activity. For convenience, as simplified gap junction is illustrated; the cartoon does not indicate that association is exclusively pre- or postsynaptic. Modified from Flores et al., 2010 [120].