Connexin hemichannel mediated ephaptic inhibition in the retina

Abstract

Connexins are the building blocks of gap-junctions; sign conserving electrical synapses. Recently it has been shown that connexins can also function as hemichannels and can mediate a sign inverting inhibitory synaptic signal from horizontal cells to cones via an ephaptic mechanism. In this review we will discuss the critical requirements for such an ephaptic interaction and relate these to the available experimental evidence. The highly conserved morphological structure of the cone synapse together with a number of specific connexin proteins and proteoglycans present in the synaptic complex of the cones creates a synaptic environment that allows ephaptic interactions. The connexins involved are members of a special group of connexins, encoded by the GJA9 and GJA10 genes. Surprisingly, in contrast to many other vertebrates, mouse and other rodents seem to lack a GJA9 encoded connexin. The specific combination of substances that block feedback and the highly specific modification of feedback in a zebrafish lacking Cx55.5 hemichannels all point to an ephaptic feedback mechanism from horizontal cells to cones.

This article is part of a Special Issue entitled Electrical Synapses.

Introduction

In the outer retina, horizontal cells feed back negatively to cones. This inhibitory pathway forms the basis for the center surround organization of bipolar cells. The underlying mechanism of this inhibitory pathway is a matter of intense debate. Three hypotheses have been put forward. One hypothesis Proposes that horizontal cells feed back to cones via a GABA-ergic pathway (Wu and Dowling, 1980, Tachibana and Kaneko, 1984, Tatsukawa et al., 2005). The second hypothesis proposes that the feedback signal from horizontal cells to cones is a modulation of the pH in the synaptic cleft (Hirasawa and Kaneko, 2003, Vessey et al., 2005) and the third hypothesis proposes that horizontal cells feed back to cones via an ephaptic feedback mechanism involving connexin hemichannels (Kamermans et al., 2001, Kamermans and Fahrenfort, 2004, Fahrenfort et al., 2009, Klaassen et al., 2011). The latter feedback pathway still functions in the presence of GABA antagonists, indicating that, at least, this part of feedback is GABA independent. In the present review, we will discuss the ephaptic mechanism and how this mechanism relates to the available experimental data.

The synaptic complex between cones, horizontal and bipolar cells is highly conserved and complex. It is characterized by synaptic ribbons and deep invaginations (Fig. 1A). The dark resting membrane potential of cones is about −40 mV. At that potential the L-type Ca²⁺-channels, located near the synaptic ribbon, are activated and Ca²⁺ will flow into the cell. This induces a constant release of glutamate. Glutamate will activate ionotropic glutamate receptors on horizontal cells and OFF-bipolar cells and metabotropic glutamate receptors on ON-bipolar cells. Light stimulation leads to hyperpolarization of cones, a reduction of their Ca²⁺-current and a reduction of glutamate release, which induces hyperpolarization of horizontal cells and OFF-bipolar cells and depolarization of ON-bipolar cells. Horizontal cells are extensively electrically coupled by gap-junctions and feed back to cones. The result of this feedback pathway is a modulation of the Ca²⁺-current of cones (Verweij et al., 1996, Pottek et al., 2003, Verweij et al., 2003, Hirasawa and Kaneko, 2003, Cadetti and Thoreson, 2006, Thoreson et al., 2008). The Ca²⁺-current is modulated in a highly specific manner. With hyperpolarization of horizontal cells, the cone Ca²⁺-current activates at more negative potentials. Such a modulation of the Ca²⁺-current leads to an increase of glutamate release by the cones. This is a negative feedback pathway. Although there is general agreement that horizontal cells feed back to cones by modulating the Ca²⁺-current (Pottek et al., 2003, Verweij et al., 2003, Hirasawa and Kaneko, 2003, Cadetti and Thoreson, 2006, Thoreson et al., 2008), the underlying mechanism is an issue of debate.

Byzov and Shura-Bura (1986) suggested that horizontal cells feed back to cones by means of an electrical mechanism. This hypothesis was based on two key notions. First, the synaptic complex is highly convoluted suggestive for a relatively high extracellular resistance. Second, the glutamate receptors of horizontal cells are continuously activated in the dark due to the sustained glutamate release by cones. These two specifics imply that in the dark current will flow into the horizontal cells via the glutamate receptors. Since this current has to come from outside the synaptic complex, it has to pass through the extracellular space in the synaptic cleft. Given the fact that the synapse is highly convoluted, this extracellular space will have a finite resistance. A current through a resistance will induce a voltage drop. The result is that the potential deep in the synaptic cleft is slightly negative. The cone Ca²⁺-channels, which are located near the synaptic ribbon, will therefore sense a more depolarized membrane potential than the actual membrane potential of the cone. In a voltage clamp experiment this will express itself as a shift of the Ca²⁺-current to more negative potentials. When horizontal cells are hyperpolarized, the current through the extracellular space will increase which results in an even larger negativity deep in the synaptic cleft. The final result is that the cone Ca²⁺-current will start to activate at even more negative potentials.

A strong prediction of this hypothesis is that blocking the glutamate gated current with 6,7-Dinitroquinoxaline-2,3 (1H,4H)-dione (DNQX) results in the dissipation of the negativity deep in the synaptic cleft and leads to a shift of the Ca²⁺-current in cones to more positive potentials. This prediction was tested by Verweij et al. (1996) and the opposite was found, i.e. application of DNQX led to a shift of the Ca²⁺-current to more negative potentials. The conclusion was that the ephaptic feedback model as described by Byzov could not account for negative feedback from horizontal cells to cones (Verweij et al., 1996, Kamermans and Spekreijse, 1999). Either the mechanism was completely different or something was missing from the original electrical feedback hypothesis of Byzov.

The missing component was an unexpected component, connexin hemichannels. In 2001, we found in collaboration with the group of Weiler that connexin hemichannels were located at the tips of horizontal cells, opposed to the cone synaptic ribbon (Fig. 1B) (Kamermans et al., 2001). Extending the electrical feedback model of Byzov with these channels, changes the model such that it can account for the experimental results found by Verweij et al. (1996). With hemichannels present in the model, application of DNQX will have a different effect on the system. DNQX will close the glutamate receptors. This will lead to hyperpolarization of horizontal cells, which will lead to an increase in the current flowing through the hemichannels and thus through the extracellular space. This will make the potential in the synaptic cleft more negative, and shift the Ca²⁺-current to more negative potentials, just as experimentally found.