Connexin 36 expression is required for electrical coupling between mouse rods and cones

Abstract

Rod-cone gap junctions mediate the so-called "secondary rod pathway", one of three routes that convey rod photoreceptor signals across the retina. Connexin 36 (Cx36) is expressed at these gap junctions, but an unidentified connexin protein also seems to be expressed. Cx36 knockout mice have been used extensively in the quest to dissect the roles in vision of all three pathways, with the assumption, never directly tested, that rod-cone electrical coupling is abolished by deletion of this connexin isoform. We previously showed that when wild type mouse cones couple to rods, their apparent dynamic range is extended toward lower light intensities, with the appearance of large responses to dim flashes (up to several mV) originating in rods. Here we recorded from the cones of Cx36del[LacZ]/del[LacZ] mice and found that dim flashes of the same intensity evoked at most small sub-millivolt responses. Moreover, these residual responses originated in the cones themselves, since: (i) their spectral preference matched that of the recorded cone and not of rods, (ii) their time-to-peak was shorter than in coupled wild type cones, (iii) a pharmacological block of gap junctions did not reduce their amplitude. Taken together, our data show that rod signals are indeed absent in the cones of Cx36 knockout mice. This study is the first direct demonstration that Cx36 is crucial for the assembly of functional rod-cone gap junctional channels, implying that its genetic deletion is a reliable experimental approach to eliminate rod-cone coupling.

Voltage-clamp recordings of light responses from wild-type and mutant mouse cone photoreceptors

“…Although previous patch-clamp recordings of cone responses have been made by inserting electrodes into the retina blindly (Cangiano et al, 2012;Asteriti et al, 2014Asteriti et al, , 2017, we thought it might be useful to develop a method to record from the photoreceptors under visual control. Although we contemplated recording from retinas with cones or cone sheaths labeled with a fluorescent dye, we were concerned that the light used to excite the dye might lightadapt the retina.…”

“…In most cones, however, this faster waveform was followed by a component of slower decay that varied in amplitude, typically ∼5 pA but as large as 15 pA. This slower component was likely to be produced by rod input entering the cone through gap junctions (Raviola and Gilula, 1975;Schnapf, 1995, 1999;Hornstein et al, 2005;Asteriti et al, 2014Asteriti et al, , 2017. Fig.…”

“…In Gnat1 −/− retinas, rodspecific transducin α is knocked out, yielding electrically silent but otherwise healthy rods (Calvert et al, 2000). Cx36 −/− cones lack the gap-junction protein that forms electrical synapses with neighboring rods (Deans et al, 2002;Asteriti et al, 2017). Cones from Gnat1 −/− and Cx36 −/− retinas recovered rapidly from brief flashes with consistently monophasic waveforms (Fig.…”

“…Much of interest has been learned about mouse cone transduction from previous measurements with suction-electrode recording (Nikonov et al, 2005(Nikonov et al, , 2006, perforated-patch voltage recording (Cangiano et al, 2012;Asteriti et al, 2014), and whole-retina isolated photoreceptor recording (for example, Sakurai et al, 2011Sakurai et al, , 2015Morshedian et al, 2019). These experiments have provided new information about the physiological significance of rod/cone coupling (Asteriti et al, 2014(Asteriti et al, , 2017 and the role of transduction proteins in producing the cone light response (Sakurai et al, 2011(Sakurai et al, , 2015. Recordings from whole Gnat1 −/− retinas lacking rod responses in the presence of synaptic and Müller-cell blockers have been particularly valuable in studying mechanisms of cone pigment regeneration Morshedian et al, 2019).…”

Molecular dissection of cone photoreceptor‐enriched genes encoding transmembrane and secretory proteins

“…Cone photoreceptors establish connections with different subtypes of cone bipolar neurons and horizontal cells for transmission and integration of visual signals (Hoon, Okawa, Della Santina, & Wong, 2014;Missaire & Hindges, 2015;Seung & Sumbul, 2014). Cones are tightly packed within the ONL and electrically coupled to rods via gap junctions, allowing the transmission of scotopic signals through the photopic pathway in low-light conditions (Asteriti, Gargini, & Cangiano, 2017; O'Brien, Chen, Macleish, O'Brien, & Massey, 2012;Tsukamoto, Morigiwa, Ueda, & Sterling, 2001). Importantly, secondary death of cone photoreceptors in retinitis pigmentosa is, in part, correlated to the loss of Rod Derived Cone Viability factor, which is secreted by rod photoreceptors and protects cone cells (Ait-Ali et al, 2015;Leveillard et al, 2004).…”