Genetic Method for Labeling Electrically Coupled Cells: Application to Retina

Abstract

Understanding how the nervous system functions requires mapping synaptic connections between neurons. Several methods are available for imaging neurons connected by chemical synapses, but few enable marking neurons connected by electrical synapses. Here, we demonstrate that a peptide transporter, Pept2, can be used for this purpose. Pept2 transports a gap junction-permeable fluorophore-coupled dipeptide, beta-alanine-lysine-N-7-amino-4-methyl coumarin-3-acid (βALA). Cre-dependent expression of pept2 in specific neurons followed by incubation in βALA labeled electrically coupled synaptic partners. Using this method, we analyze light-dependent modulation of electrical connectivity among retinal horizontal cells.

Keywords: PEPT2; electrical synapse; gap junction; horizontal cells; peptide transporter; retina.

FIGURE 1

Schematic illustration of genetic method for labeling electrically coupled cells from Cre-positive cells. A channel or transporter is expressed in Cre-positive cells, allowing them to take up gap junction permeable fluorescent substrates. The substrates diffuse through gap junctions to label coupled cells.

FIGURE 2

Pept2 method enables labeling gap junction-coupled cells. (A) Cultured HEK cells stained with antibody against connexin 43 (Cx 43). Dapi shows cell nucleus and Neurotrace stains cytoplasm. Connexin 43 is enriched at the cell membrane between neighboring cells. (B) HEK cells injected with neurobiotin and stained with Texas red-streptavidin. Red arrow indicates the injected cell. (C) HEK cells injected with βALA. Red arrow indicates the injected cell. (D) Schematic illustration of the method for testing channels or transporters capable of importing small fluorescent substrate that can permeate gap junctions. (E) HEK cells were transfected with GFP and Pept2, then mixed with a 100-fold excess of untransfected cells and incubated with βALA. βALA diffused from GFP positive cells to adjacent coupled cells. (F) HEK cells were transfected with GFP, GFP + Pept2 or Pept2-GFP. Uptake of βALA was eliminated by Pept2 inhibitor Gly–Gln. Scale bars in (A–C,E): 20 μm; in (F): 100 μm.

FIGURE 3

Both Pept2-GFP and Pept2-p2a-GFP mediate βALA uptake. HEK cells were transfected with Pept2-GFP (A) or Pept2-p2a-GFP (B). Uptake of βALA was blocked by Pept2 inhibitor Gly–Gln. Diffusion of βALA to coupled cells was blocked by gap junction blockers MFA and CBX. Scale bar: 20 μm.

FIGURE 4

Pept2 method enables quantification of gap junction strength between cultured cells. (A) Schematic illustration and measurement of the βALA signals for quantification of gap junction strength. (B) βALA signals from the probe cell to coupled cells follow an exponential decay. (C) Inhibition of βALA diffusion between gap junction-coupled cells by MFA at indicated concentrations. (D) Inhibition of βALA diffusion between gap junction-coupled cells by CBX at indicated concentrations.(E) Decay curves fitted from results in (C).(F) Decay curves fitted from results in (D). (G) Coupling strength S calculated based on curves in (E). ^∗p < 0.05; ^∗∗p < 0.01, n = 5 probe cells for each condition. Error bars are SEM. (H) Coupling strength S calculated based on (F). ^∗p < 0.05; ^∗∗p < 0.01, n = 5 probe cells for each condition. Error bars are SEM. Scale bars in (C,D): 20 μm.

FIGURE 5

Pept2 dependent method reveals coupled cells in the retina. (A) Schematic of Cre-dependent AAV carrying Pept2-GFP. (B) βALA signals were bright in Pept2-GFP positive J-RGCs in the ganglion cell layer (GCL), and also present in coupled amacrine cells in the INL. (C) Diffusion of βALA to coupled amacrine cells was eliminated by MFA. Scale bars in (B,C): 40 μm. (D) Pept2 method reveals coupling of horizontal cells. (E) Diffusion of βALA to coupled cells was eliminated by MFA. Scale bars in (D,E): 20 μm. Asterisk indicates a blot on the cover slide.

FIGURE 6

Analysis of light dependent electrical coupling between horizontal cells. (A) Coupling between horizontal cells is stronger under dim light than under bright light. Horizontal cells are identified by the marker calbindin (Calb). Blood vessels are also stained by the secondary antibody against mouse Calb primary antibody, but can be easily distinguished from horizontal cells. (B) Quantification of electrical coupling strength S in (A). ^∗∗p < 0.01, n = 73 starter cells in bright light, n = 45 starter cells in dim light. Error bars are SEM. (C) Schematic of assays to measure gap junction strength between horizontal cells under local stimulus in intact retina (step1 → step2). For cutting experiments, retinas were cut before incubation (step2 → step1). (D) Coupling strength of illuminated horizontal cells in a ∼4 mm² window (IN) and those in an adjacent unilluminated region (OUT). (E) Quantification of gap junction strength from 6 separate retinas treated as in (D). n = 5–19 starter cells for each condition per retina. Error bars are SEM. (F) Coupling strength of illuminated horizontal cells in a ∼4 mm² window (Cut_IN) and those in an adjacent unilluminated region (Cut_OUT) from cut retina. (G) Quantification of gap junction strength from 3 separate retinas treated as in (F). n = 5–19 starter cells for each condition per retina. Error bars are SEM. Scale bars in (A,D,E): 20 μm.