Physiological properties of rod photoreceptor electrical coupling in the tiger salamander retina

Abstract

Using dual whole-cell voltage and current clamp recording techniques, we investigated the gap junctional conductance and the coupling coefficient between neighbouring rods in live salamander retinal slices. The application of sinusoidal stimuli over a wide range of temporal frequencies allowed us to characterize the band-pass filtering properties of the rod network. We found that the electrical coupling of all neighbouring rods exhibited reciprocal and symmetrical conductivities. On average, the junctional conductance between paired rods was 500 pS and the coupling coefficient (the ratio of voltage responses of the follower cell to those of the driver cell), or K-value, was 0.07. Our experimental results also demonstrated that the rod network behaved like a band-pass filter with a peak frequency of about 2-5 Hz. However, the gap junctions between adjacent rods exhibited linearity and voltage independency within the physiological range of rods. These gap junctions did not contribute to the filtering mechanisms of the rod network. Combined with the computational modelling, our data suggest that the filtering of higher frequency rod signals by the network is largely mediated by the passive resistive and capacitive (RC) properties of rod plasma membranes. Furthermore, we found several attributes of rod electrical coupling resembling the physiological properties of gene-encoded Cx35/36 gap junctions examined in other in vitro studies. This indicates that the previously found Cx35/36 expression in the salamander rod network may be functionally involved in rod-rod electrical coupling.

Figure 1. Rod photoreceptors and their expression of connexin 35/36

A, Cx35/36 (green) immunoreactivity in the salamander outer retina. Vertical single optical section focusing at the outer nuclear layer (ONL). The retina was double labelled with recoverin antibodies (red). Rods (R, faintly stained) were located in the upper tier, whereas cones (C, strongly stained) were located in the lower tier. The characteristic round and elongated Cx35/36-positive plaques were found between rod somas in the distal portion of the ONL (arrows). Scale bar = 10 μm. B, visualization of a pair of rods simultaneously patch clamped and filled with Lucifer yellow through two recoding pipettes.

Figure 2. Simultaneous dual whole-cell voltage clamp recordings from a pair of neighbouring salamander rods in the slice preparation

A, the two paired rods were voltage clamped at −40 mV. When a series of voltage step commands (V₁) were applied to cell1 (driver cell), the voltage-activated current responses (I₁) were recorded in cell 1 (left panel), and the junctional currents of the opposite polarity (I₂) were recorded in cell 2 (follower cell, right panel). B, the plot of transjunctional current (I_j) as a function of transjunctional voltage (V_j) at the steady state (•) seen in A. C, distribution of junctional conductance for 28 rod–rod pairs. D, the plot of the junctional conductance in each direction. G_j1,2 represents the junctional conductance measured from cell 1 coupled to cell 2, whereas G_j2,1 represents the junctional conductance measured from cell 2 coupled to cell 1. The diagonal line indicates the expected value, showing that the junctional conductance in either direction is similar.

Figure 3. Simultaneous dual whole-cell current clamp recordings from a pair of neighbouring rods

A, when a series of current step commands (I₁) were applied to cell 1, the voltage responses (V₁) were recorded in cell 1 (left panel) and the evoked voltage responses (V₂) of the same polarity were recorded in cell 2 (right panel). B, the plots of voltage responses of cell 1 and cell 2 corresponding to V₁ and V₂, respectively, in an expanded time scale. C, the plots of voltage responses of V₁ and V₂ as a function of currents injected into the driver cell (I₁) at the instantaneous state (filled symbols) and the steady state (open symbols). D, the plot of the coupling coefficient in each direction of paired rods. The diagonal line indicates the expected value, showing that the coupling coefficient in either direction is similar. E, the plot of the K ratio versus the ratio of the rod input resistance (see text). The diagonal line indicates the expected value, showing that the K ratio is similar to the ratio of the input resistance.

Figure 4. The voltage-dependent properties of the rod network

A, the effect of varying membrane potentials on the rod electrical transmission. When the membrane potential of cell 2 was set at different voltages by injecting varying currents (I₂), a sinusoidal current of 5 Hz (I₁) applied to cell 1 elicited voltage responses in cell2 (V₂). B, the plot of normalized coupling coefficient (K) (to that determined at 0 pA holding current) at each given I₂. The curve was fitted with the Gaussian equation. C, the effect of varying membrane potentials on rod junctional conductance. A series of voltage step commands applied to cell 1 (V₁) activated junctional currents in cell 2 (I₂) at different holding potentials (V_{2 h}: 0 mV, −40 mV and −80 mV). D, the plot of the I_j–V_j relation at each given holding potential. The conductance was determined by the slope of the I_j–V_j curve. E, the plot of the normalized conductance (to that determined at −40 mV holding potential) as the function of the holding potential of cell 2. The curve was fitted with the Boltzmann equation. The holding potential that produced half conductance was estimated at −2.66 mV.

Figure 5. The frequency-dependent properties of the rod network

A, the sinusoidal current stimuli (I₁) of varying frequencies applied to cell 1 elicited voltage responses in cell 1 (V₁, grey curve) and in cell 2 (V₂, black curve). B, the plots of the normalized V₁ (Δ), V₂ (▴), K (•) (left axis), and the degree of phase shift (O, right axis) as the function of current frequency. The cut-off frequency was estimated as 33 Hz. C, the sinusoidal voltage stimuli (V₁) of varying frequencies applied to cell1 activated transjunctional currents in cell 2 (I₂). D, the plot of the normalized transjunctional current (I/I_max) as the function of voltage frequency.

Figure 6. The computer modelling of the rod network

A, a schematic diagram of the equivalent circuit of the rod pair. The two next neighbouring rods are interconnected by a resistance of R_j (= 1/G_j). R₁, R₂ and C represent the lumped equivalent of the resistance and capacitance across each rod membrane in the pairs, where R₁= 1/G₁ and R₂= 1/G₂ (G₁ and G₂ are the conductance of cell 1 and cell 2, respectively). B, the computer simulated normalized coupling coefficient (K_N) changes as a function of frequency (when G_j and R₂ were manipulated using different values). C, the plot of cut-off frequency (see eqn (3) in Results) as the function of junctional conductance at each given membrane input resistance. The calculated cut-off frequency is 32 Hz (*) when G_j, R₂, and C are 500 pS, 250 MΩ, and 40 pF, respectively.

Figure 7. The intensity dependence of the coupled rod network

The plots of the voltage-intensity relation of the driver cell (Δ), the follower cell without considering filtering properties of the rod network (•), and the follower cell having peak responses filtered by the rod network (O) as light intensity increases.