Heterocellular Coupling Between Amacrine Cells and Ganglion Cells

All superclasses of retinal neurons, including bipolar cells (BCs), amacrine cells (ACs) and ganglion cells (GCs), display gap junctional coupling. However, coupling varies extensively by class. Heterocellular AC coupling is common in many mammalian GC classes. Yet, the topology and functions of coupling networks remains largely undefined. GCs are the least frequent superclass in the inner plexiform layer and the gap junctions mediating GC-to-AC coupling (GC::AC) are sparsely arrayed amidst large cohorts of homocellular AC::AC, BC::BC, GC::GC and heterocellular AC::BC gap junctions. Here, we report quantitative coupling for identified GCs in retinal connectome 1 (RC1), a high resolution (2 nm) transmission electron microscopy-based volume of rabbit retina. These reveal that most GC gap junctions in RC1 are suboptical. GC classes lack direct cross-class homocellular coupling with other GCs, despite opportunities via direct membrane contact, while OFF alpha GCs and transient ON directionally selective (DS) GCs are strongly coupled to distinct AC cohorts. Integrated small molecule immunocytochemistry identifies these as GABAergic ACs (γ+ ACs). Multi-hop synaptic queries of RC1 connectome further profile these coupled γ+ ACs. Notably, OFF alpha GCs couple to OFF γ+ ACs and transient ON DS GCs couple to ON γ+ ACs, including a large interstitial amacrine cell, revealing matched ON/OFF photic drive polarities within coupled networks. Furthermore, BC input to these γ+ ACs is tightly matched to the GCs with which they couple. Evaluation of the coupled versus inhibitory targets of the γ+ ACs reveals that in both ON and OFF coupled GC networks these ACs are presynaptic to GC classes that are different than the classes with which they couple. These heterocellular coupling patterns provide a potential mechanism for an excited GC to indirectly inhibit nearby GCs of different classes. Similarly, coupled γ+ ACs engaged in feedback networks can leverage the additional gain of BC synapses in shaping the signaling of downstream targets based on their own selective coupling with GCs. A consequence of coupling is intercellular fluxes of small molecules. GC::AC coupling involves primarily γ+ cells, likely resulting in GABA diffusion into GCs. Surveying GABA signatures in the GC layer across diverse species suggests the majority of vertebrate retinas engage in GC::γ+ AC coupling.

Introduction

Retinal ganglion cells (GCs) are the signal outflow cells of the vertebrate retina: a network layer that integrates bipolar cell (BC) and amacrine cell signals and passes them to CNS targets. Like BCs, most GCs are part of a unidirectional synaptic chain, not evidencing any direct feedback to the preceding input stage. However, early physiological studies established the ability of a GC to excite amacrine cells, other GCs and even itself (Matsumoto, 1975; Marchiafava, 1976; Marchiafava and Torre, 1977; Mastronarde, 1983; Sakai and Naka, 1988; Sakai and Naka, 1990). This excitation was always sign-conserving and occurred with short latency, yet electrical synaptic transmission was often dismissed due to a lack of anatomical evidence, in stark contrast to many other retinal neurons (Vaney, 1994). Later, intracellular biotinylated tracer injection studies (Vaney, 1991, 2002; Vaney and Weiler, 2000) showed tracer diffusion patterns between ganglion and amacrine cells that were interpreted as coupling mediated by gap junctions (e.g., Bloomfield and Xin, 1997; Massey, 2008), and more recently confirmed with gap junction protein knockout mice (e.g., Schubert et al., 2005a,b; Pan et al., 2010).

Gap junctions are intercellular channels that mediate the flux of small molecules and ions and, therefore, are the anatomical basis for electrical synaptic transmission in the nervous system. Like chemical synapses, gap junctions are extremely diverse structures mediating intercellular signaling. The primary proteins of gap junctions are drawn from a large family of connexins with four transmembrane spanning domains, cytosolic domains that usually (though not always) provide predominantly homotypic or bihomotypic binding even if the junctions are heteromeric (Li et al., 2008; Rash et al., 2013), and intracellular domains that mediate recognition and binding of other gap junction proteins. In general, it is thought that the peak open conductance of a single connexon is principally related to its pore diameter (this is not always true) with complex modulation enabled by a range of mechanisms (Ek-Vitorin and Burt, 2013; Hervé and Derangeon, 2013) including connexin phosphorylation (Pereda et al., 2013; O’Brien, 2017), methanesulfonate-analog (taurine) binding (Locke et al., 2011), and many different adapter protein interactions (e.g., Zou et al., 2017). Light-induced changes in gap junctions are currently understood to modify the open conductance of a connexon through these mechanisms, but will not change the presence or absence of gap junctions at contact sites with coupling partners. That said, photopic or scotopic changes may alter gap junctional sizes.

Modes of coupling in the retina can be grouped into broad categories such as homocellular (coupling between the same “types” of cells) and heterocellular (coupling between different cell types). But what do we mean by “type” in the context of retina? Our terminology is based on computational classification theory where a class