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1 Cullen Eye Institute, Baylor College of Medicine, One Baylor Plaza, NC-205, Houston, TX 77030, USA
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(Received 8 January 2005;
accepted after revision 25 February 2005;
first published online 3 March 2005)
Corresponding author J. Zhang: Cullen Eye Institute, Baylor College of Medicine, One Baylor Plaza, NC-205, Houston, TX 77030, USA. Email: jianz{at}bcm.tmc.edu
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Introduction |
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The physiological properties of rod electrical coupling have been studied extensively in the salamander retina by Attwell and colleagues (Attwell & Wilson, 1980; Attwell et al. 1984), who demonstrated that injecting a –1 nA current into a rod evoked a hyperpolarization of about 20 mV in an adjacent rod and about 4 mV in an adjacent cone. This suggests that rods are strongly electrically coupled to neighbouring rods and are weakly coupled to neighbouring cones. These data, in conjunction with computer simulations of the photoreceptor network, led to the estimation of a coupling resistance of 300 M
between adjacent rods and a resistance of 5000 M between adjacent rods and cones, with the assumption that each rod was electrically coupled to four neighbouring rods and four neighbouring cones (Attwell et al. 1984). It is still not clear, however, what type(s) of gap junctions are present between rods or what the biophysical properties of rod gap junctions would be in transmitting rod signals laterally from one rod to its adjacent rods.
The coupled rod network has distinct temporal characteristics. In the turtle retina, Detwiler et al. (1978, 1980) first showed that the time-to-peak responses following the onset of a light flash was shorter in rods further away from a bar of light, indicating that the rod network behaves like a high-pass filter. By introducing an ‘inductive’ pathway in their model, several studies suggested that the activation of a voltage- and time-dependent inward rectifying current (Ih) (Attwell & Wilson, 1980; Hestrin, 1987) might be responsible for the rod high-pass filtering property (Detwiler et al. 1978, 1980; Torre & Owen, 1983; Attwell et al. 1984). Other studies, however, suggest that rod responses are band-pass filtered during light transmission (Attwell, 1986; Armstrong-Gold & Rieke, 2003), presumably due to the membrane capacitive property (Attwell, 1986). Nevertheless, it is uncertain whether the gap junctions between two adjacent rods would contribute to the filtering properties of the rod network, and therefore attenuate the kinetics of rod signals. Recent studies in other retinal neurones and in the neurones in other parts of the central nervous system (CNS) indicate that such a coupling pathway may behave as a low pass filter (Veruki & Hartveit, 2002a,b; Nolan et al. 1999; Galarreta & Hestrin, 2001).
Our previous study investigated the cellular localization of gap junction proteins in the salamander retina with a fish connexin35 (Cx35) antibody (that also recognizes murine connexin36, and thus named Cx35/36 in this study). Cx35 was first cloned from the fish retina (O'Brien et al. 1996), and later a homologous murine Cx36 was identified (Condorelli et al. 1998; Söhl et al. 1998) and localized in the mammalian retina (Feigenspan et al. 2001; Mills et al. 2001; Deans et al. 2002). We found that Cx35/36 was localized to rod photoreceptors in the salamander retina (Zhang & Wu, 2004), where the ultrastructure of gap junctions has been identified (Custer, 1973; Mariani, 1986). Nonetheless, whether the biophysical profiles of rod electrical coupling agree with the physiological properties of Cx35/36 examined in in vitro systems (White et al. 1999; Srinivas et al. 1999; Teubner et al. 2000) is unknown. Thus it is of great interest to compare the physiology of salamander rod electrical coupling with the Cx35/36 gap junctional properties identified in in vitro (Srinivas et al. 1999; White et al. 1999; Teubner et al. 2000).
In order to elucidate the biophysical properties of rod electrical coupling, we used the dual whole-cell voltage- and current-clamp recording techniques to directly measure the junctional conductance and the coupling coefficient between paired rods in tiger salamander retinal slices, and also to determine at quantitative levels the strength and dynamics of rod–rod coupling. Here we show that the conductivity of amphibian rod gap junctions is smaller than previously estimated with an average conductance of about 500 pS and an average coupling coefficient (K) of 0.07. Our experimental results, combined with the modelling of the electrically coupled rod network, also suggest that rods behave like a band-pass filter and that the gap junctions between rods do not contribute to the filtering mechanisms of the rod network. In addition, we also compared several attributes of rod electrical coupling with the physiological properties of gene-encoded Cx35/36 gap junctions examined in other in vitro studies.
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Methods |
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Larval tiger salamanders (Ambystoma tigrinum) purchased from Charles D. Sullivan, Co. (Nashville, TN, USA) and KON's Scientific Co. Inc. (Germantown, WI, USA) were used in this study. The University Committee on Animal Use at Baylor College of Medicine approved the use of animals and all animals were treated in accordance with the NIH guidelines. The dissection procedures have been previously described (Werblin, 1978; Wu, 1987). In brief, the animals were maintained on a daily 12-h light–dark cycle. The animals were decapitated and the eyes were enucleated and hemisected. The cornea, lens and vitreous were carefully removed. The retina was then removed from the posterior eyecups and was flattened, photoreceptor side up, on filter paper. The retina and filter paper were sectioned into 200–300 µm thick slices. Slices were maintained in Ringer solution at room temperature (22°C).
Dual whole-cell recordings
The extracellular Ringer solution consisted of (mM): 111 NaCl, 2.5 KCl, 1.8 CaCl2, 1 MgCl2, 10 dextrose, and 5 Hepes, and the pH was adjusted to 7.8 with NaOH. In some experiments, 40 mM tetraethyl ammonium chloride (TEA-Cl) replaced equimolar NaCl in the extracellular medium to block voltage-dependent outward-rectified Ik. There was no significant difference in the averaged gap junctional conductance between the two groups (with or without TEA). The electrodes were pulled from borosilicate glass (TW150F-4, World Precision Instruments, Sarasota, FL, USA) using a Flaming/Brown P-87 puller (Sutter Instrument, Novato, CA, USA), and they had a resistance of 5–7 M when filled with an intracellular solution containing the following (mM): 106 potassium gluconate, 5 NaCl, 2 MgCl2, 5 EGTA and 5 Hepes, adjusted to pH 7.4 with KOH.
The experiments were performed in dim room lighting. Dual whole-cell recordings were obtained from paired salamander rods with an EPC9/2 amplifier (HEKA Elektronik, Lambrecht, Germany) in voltage- or current-clamp mode. Most of the pipette capacitance was neutralized in the cell-attached configuration using the ‘Cfast’ capacitance neutralization network built into the EPC-9. After the whole-cell configuration was established, the membrane potential of both rods was initially held at –40 mV. Electrical coupling between rods was measured by applying a series of voltage or current step commands to one rod (the driver cell) in order to elicit a membrane current or voltage response in the adjacent rod (the follower cell). Current and voltage signals were filtered at 3 kHz and digitized at 2–5 kHz using a Pentium computer equipped with an ITC-16 data acquisition board (HEKA Elecktronik). Pulse software (v8.65, HEKA Elecktronik) was used to generate voltage and current commands.
Data analysis was carried out using PulseFit (v8.65, HEKA Elecktronik), Igor Pro (v4.08, WaveMetrics, Lake Oswego, OR, USA), Excel 2000 (Microsoft), and Origin (v7.0, OriginLab Corp., Northampton, MA, USA). The junctional conductance (Gj) and the coupling coefficient (K) were estimated by calculating the slope of the linear regression curve used to fit the original data. The equations were defined as follows:
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| (1) |
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V=Vf–Vd, Vf being the membrane voltage of the follower cell, and Vd being the membrane voltage of the driver cell). All statistic values were given as the mean ± standard deviation and n is the number of cells unless elsewhere indicated. Immunocytochemistry
The salamander retinas were fixed in fresh 4% paraformaldehyde/phosphate buffered saline (PBS), pH 7.8, for 30–60 min at room temperature. Following fixation, they were rinsed extensively with PBS. For double-label experiments, the pieces of free-floating vibratome sections were blocked with 3% donkey serum in PBS with 0.5% Triton X-100–0.1% sodium azide for 2 h to overnight in order to reduce non-specific labelling. The tissues were then incubated in a mixture of primary antibodies in the presence of 1% donkey serum–PBS–0.5% Triton X-100–0.1% sodium azide for 3–5 days at 4°C. Controls lacking primary antibodies were blank. Following extensive washes with PBS containing 0.5% Triton X-100–0.1% sodium azide, immunoreactivity was revealed by overnight incubation with immunofluorescent secondary antibodies conjugated to appropriated fluorochromes. After extensive rinsing, the tissues were mounted with Vectashield (Vector Laboratories, Inc., Burlingame, CA, USA) and observed with a confocal laser-scanning microscope (Zeiss LSM 510, Carl Zeiss, Inc., Thornwood, NY, USA). Images were acquired using a 40x or 63x oil-immersion objective lens and Zeiss LSM-PC software. Intensity and size of the images were adjusted using Adobe Photoshop (v 5.0).
A mouse monoclonal antibody against Cx35/36 (clone 8F6.2) was obtained from Chemicon International (Temecula, CA, USA). It was used at a dilution of 1: 1000 for immunocytochemistry. A rabbit polyclonal antibody against bovine recoverin (1: 1000) was kindly provided by Dr A. M. Dizhoor (Pennsylvania College Optometry, Elkins Park, PA, USA). Secondary antibodies used in the experiments were donkey antimouse IgG and donkey antirabbit IgG conjugated to CY3 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) or Alexa 488 (Molecular Probes, Eugene, OR, USA) and used in the dilution of 1: 100 in PBS containing 0.5% Triton X-100–0.1% sodium azide.
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Results |
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Our previous study has demonstrated the occurrence of Cx35/36 gap junctions in rod photoreceptors of the tiger salamander retina (Zhang & Wu, 2004). In the rod sublayer (red, labelled by recoverin antibodies) of the outer nuclear layer (ONL), Cx35/36-positive plaques (green, arrows) are present between rod cell bodies (Fig. 1A), and are restricted at the level distal to the external limiting membrane (Mariani, 1986). The plaques are colocalized with the rod fins where the ultrastructure of gap junctions was observed (Custer, 1973; Mariani, 1986; Zhang & Wu, 2004). Therefore, in order to characterize the biophysical properties of rod gap junctions, all recordings were restricted to the rod sublayer. In some cases, Lucifer yellow dye was loaded into the cells through two patch pipettes to further identify the morphology and position of the rods. One such example is shown in Fig. 1B.
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Rod–rod junctional conductance
Simultaneous patch clamp recordings were obtained from a total of 38 pairs of rods. The mean input resistance, measured in the linear region of the I–V relationship (between –40 to –35 mV), was 275 ± 22 M (n= 71). To examine the direct current flow through the electrical synapses of paired rods, we first set the whole-cell configuration in the voltage clamp mode. The membrane potential of two adjacent rods was initially held at –40 mV, near their dark membrane potential (Attwell & Wilson, 1980). A voltage step series (V1) (from –120 to 40 mV with an increment of 20 mV) applied to one rod (driver cell) evoked current responses (I2) in the neighbouring rod (follower cell) of opposite polarity to the responses in the driver cell (I1) (Fig. 2A). This observation confirmed that the rod–rod coupling was preserved in the slice preparations. The appearance of opposite polarity of the transjunctional currents illustrated the presence of a sign-conserving electrical synapse between rod photoreceptors. The relation of transjunctional current (Ij) (measured in the follower cell) and transjunctional voltage (Vj, see Methods) at the steady state (•, Fig. 2A) was approximately linear at the membrane potentials tested (Fig. 2B). Switching the driver/follower cell positions resulted in similar current responses and linear relations of Ij–Vj. This property was observed in all pairs of next-neighbouring rods.
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The coupling coefficient of rod–rod electrical synapses
The coupling coefficient (K) of rod–rod electrical synapses can be measured by determining the ratio of the voltage responses of the neighbouring follower cell to that of the driver cell, as shown in the rat retinal studies by Veruki & Hartveit (2002a,b). In the current clamp configuration, injecting a series of negative currents (I1, from –1000 to –100 pA with the increment of 150 pA) into a driver cell evoked a fast transient hyperpolarization followed by a characteristic peak-to-plateau depolarization (V1) in the driver cell (Fig. 3A). The voltage responses in the follower cell (V2) exhibited the same polarity as those measured in V1 (Fig. 3A). Injecting a series of positive currents (from 50 to 650 pA with the increment of 150 pA) into the same driver cell revealed a depolarization in the driver cell and a smaller depolarization in the follower cell (Fig. 3A). Note that the voltage responses of the follower cell exhibited a transient hyperpolarization, which was similar to those of the driver cell. However, the temporal property of the follower cell's transient voltage responses was slower and broader than that of the driver cell (Fig. 3B). The time-to-peak responses of the follower cell (varied with the currents injected into the driver cells) were about 20–100 ms slower than those of the driver cell. Switching the driver/follower cell positions did not change the voltage responses of the driver/follower cells. The activity observed in all pairs of next-neighbouring rods was similar. Measuring the amplitude of the voltage responses at the onset (filled symbols) or the offset (open symbols) of the current injection steps also revealed a rectified relationship between the currents injected to the driver cell and the voltage responses of the driver and follower cells at more depolarizing potentials (Fig. 3C).
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The voltage independency of rod–rod electrical transmission
The measurement of the gap junctional conductance demonstrated that the rod–rod coupling conductivity exhibited weak voltage sensitivity within the physiological range of rod photoreceptors (see Fig. 2B). This implies that the reciprocal communication between rods is independent of the transjunctional voltage, especially when rods are illuminated uniformly in the dark when their membrane potential is usually at –40 mV. But it is not known whether the efficient transmission between rods is affected when the resting membrane potentials of two rods differ. To test this idea, two adjacent rods were current clamped at 0 pA. As shown in Fig. 4A, a sinusoidal current pulse of 5 Hz (I1) applied to the driver cell evoked a peak-to-peak voltage response (V2) of 60 mV in the follower cell at its resting membrane potential. When the follower cell resting membrane potential was moderately hyperpolarized or depolarized by injecting a small negative or positive current, the amplitude of the follower cell voltage responses elicited by the current injection to the driver cell was less affected. However, when the resting membrane potential of the follower cell was either hyperpolarized to about –70 mV or depolarized to about –10 mV by injecting a current of ± 280 pA (I2), and the same sinusoidal current pulse of 5 Hz was applied to the driver cell, the amplitude of the follower cell voltage responses was partially suppressed. As the follower cell was hyperpolarized to more than –100 mV or depolarized to more than 0 mV, the same sinusoidal current pulse applied to the driver cell elicited much less voltage responses in the follower cell. When the normalized coupling coefficient (K) (to that determined at 0 pA holding current) was plotted as a function of the currents injected into the follower cell (Fig. 5B) and was fitted by the Gaussian curve, it showed a bell-shaped relation, reflecting that within a certain range of current injections (between –300 and 50 pA), the normalized K remained relatively constant. Outside this range, the normalized K decreased. Yet, a large amount of the residual gap junctional current still remained. The same observation was found in three out of three rod pairs.
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The characteristics of frequency dependence of the rod network and gap junctions
It has been suggested that the coupled rod network acts as a band-pass filter in shaping the visual signal transmitting to the second-order cells (Attwell, 1986; Armstrong-Gold & Rieke, 2003). Yet it is not clear if the coupling pathway (or gap junctions) between rods contributes to the filtering properties of the rod network. We therefore recorded rod pairs in a whole-cell configuration that was set in the current clamp mode as shown in Fig. 5A. The sinusoidal current stimuli (I1) of varying frequencies (1–100 Hz) applied to the driver cell evoked the voltage responses of the driver cell (V1, grey curve) and the follower cell (V2, black curve). As illustrated in Fig. 5B, averaged from five rod pairs, we found that the normalized voltage response of the driver cells (V1, open triangles) was 94%, 100%, and 96% at 1 Hz, 2 Hz, and 5 Hz, respectively, whereas the normalized voltage response of the follower cell (V2, filled triangles) was 91% at 1 Hz, 100% at 2 Hz, and 87% at 5 Hz. When the frequency of the sinusoidal current stimuli (I1) increased continuously, the normalized voltage responses of both the driver and the follower cells decreased, with the latter one decreasing significantly. Similarly, the normalized coupling coefficient KN (filled circles) was 94% at 1 Hz, 100% at 2 Hz and 88% at 5 Hz, and it gradually reduced in amplitude and developed a phase-shift (open circles) over the frequency range of 5 to 100 Hz (Fig. 5B). Thus, at the low frequency (such as at 1 Hz), voltage signals of both the driver and follower cell were attenuated, and then they peaked at a frequency of about 2 Hz. In contrast, the high frequency voltage signals passing through gap junctions to the follower cell were also largely attenuated with the cut-off frequency (defined as the frequency that produced the half-maximal KN value) measured at 33 Hz. Since the attenuation of voltage responses over a wide range of temporal frequency was found not only in the follower cell but also in the driver cell, it is implied that rod voltage responses are shaped before they are transmitted to adjacent rods.
To further determine the frequency dependence of rod gap junctions, we voltage clamped the membrane potential of the follower cell (V2) at –40 mV and manipulated the membrane potential of the driver cell (V1) by applying sinusoidal voltage pulses of varying frequency. As illustrated in Fig. 5C, when the frequency of sinusoidal voltage stimuli (V1) was increased, the amplitude of the current (I2) passing through gap junctions to the follower cell did not significantly decrease over the frequency range of 1–50 Hz. Averaged from five rod pairs, we observed that, at the frequency of 50 Hz, the normalized current response was reduced by only 10 ± 3% of the maximum current measured at the frequency of 1 Hz (Fig. 5D). In contrast, the normalized KN at the frequency of 50 Hz was reduced by 57 ± 10% of the maximum KN determined at the frequency of 2 Hz. Therefore, the measurement of current responses as a function of the frequency was in direct contradiction to the measurement of voltage responses as a function of the frequency. This suggests that the higher frequency filtering property observed here most likely reflects the RC property of the rod network.
In order to confirm whether rod gap junctions behave as a resistor, and to examine how RC properties of the rod membrane are related to the filtering properties, we analysed a simplified model of the rod network (Fig. 6A). In this model, the membrane resistance (R1 (or 1/G1) and R2 (or 1/G2)) and capacitances (C) represent the lumped equivalent of the resistance and capacitance across driver (1) and follower (2) rod membranes, while two rods are connected through a resistive pathway Rj (= 1/Gj). Assuming Ih is ignored, and therefore the high pass filtering pathway is not considered here, the equation defining the relationship of coupling coefficient (K) as the function of frequency can be expressed as
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(obtained from this study), the amplitude of rod signals spreading to the adjacent rods is frequency dependent (Fig. 6B). The cut-off frequency calculated from this model is 32 Hz (*, Fig. 6C), which is very close to what we measured experimentally. In addition, considering the rod input resistance variation from 100 to 500 M, the normalized voltage responses at a given higher frequency (to the steady state responses) would be attenuated significantly. The gap junctional conductance affecting the frequency-dependent voltage attenuation would be much less spectacular than that of rod membrane input resistance (Fig. 6B and C). Therefore, the agreement of the physiological data and the mathematical calculations supports the notion that rod gap junctions behave in a linear (ohmic) manner in mediating rod–rod electrical coupling. The frequency-dependent attenuation of rod voltage responses is not surprising given the rod plasma membrane capacitive and resistive properties.
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In the salamander retina, rods are able to respond to light ranging in intensity from a few photons to several thousands of photons with characteristic temporal kinetics (Bader et al. 1978; Attwell et al. 1984; Yang & Wu, 1997). While rods respond to dim light with a slow graded hyperpolarization, their responses to brighter light become faster with a typical transient hyperpolarization. It is likely that when rod signals propagate through the rod network, the amplitude of rod voltage responses would not only be limited by the coupling strength of the electrical synapses, but would also be attenuated by the filtering mechanism as the light intensity increases. In order to determine the degree of filtering-induced attenuation at different log units of intensity, we measured the time-to-peak of rod transient responses obtained from intracellular recordings (n= 3) (see Fig. 3, Yang & Wu, 1997; and X. L. Yang & S. M. Wu, unpublished results), and calculated the amplitude of transient voltage responses of the follower cell with/without considering RC properties of the rod membrane. We found that during the brightest light flash, the rise time of this transient response could be less than 50 ms. Our calculations showed that over eight log units of the light intensity span, the dim light responses (log I < (4)) of rods with slow kinetics would efficiently pass through rod gap junctions (•, Fig. 7). However, as light intensity increases, the transient rod responses would be brief enough to be filtered (Fig. 5). Therefore, the peak amplitude of rod responses (log I > (4)) would likely be attenuated (
, frequency, Fig. 7). Thus the light responses of rods laterally spreading within the network would be intensity dependent.
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Discussion |
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Since we have previously demonstrated that Cx35/36 gap junction proteins are present in an organized pattern in the rod network of the salamander retina (Zhang & Wu, 2004), one aim of this continuing study is to understand whether the physiological property of rod electrical coupling is consistent with the profiles of Cx35/36 gap junctions. Although, to date, the lack of potent and specific Cx35/36 gap junction blockers makes it difficult to pharmacologically distinguish Cx35/36 from other connexin proteins in salamander rods, there are several lines of evidence suggesting that Cx35/36 may play an important role in the rod electrical coupling of this species. First, morphologically, we have shown that among all of the connexin proteins examined, only Cx35/36 is exclusively localized in rod photoreceptors at locations where ultrastructure of gap junctions has been identified (Custer, 1973; Mariani, 1986; Zhang & Wu, 2004). Secondly, the staining pattern of Cx35/36 in the rod network is similar to that of Cx35/36 gene-encoded channels expressed in transfected human HeLa cells (Teubner et al. 2000).
Thirdly, in the present electrophysiological study, we have demonstrated that the biophysical properties of rod gap junctions resemble the physiology of gene-encoded Cx35/36 gap junctions. Several earlier reports have shown that Cx35/36 gap junctions studied in in vitro expression systems exhibit three characteristics: (1) small channel conductance; (2) voltage independence; and (3) formation of homologous channels (Srinivas et al. 1999; White et al. 1999; Teubner et al. 2000). Similarly, in the salamander retina, the rod coupling exhibited the smaller conductance of 500 pS. Within the rod physiological range, the conductivity of rod gap junctions was independent of the transjunctional voltage between rods, and was also independent of the rod plasma membrane potential. Furthermore, since current flow passed equally well in both directions, this suggests that rod electrical synapses were symmetrical and bi-directional. These are results consistent with the notion that Cx35/36 forms homologous gap junctions between neighbouring neurones (White et al. 1999; Teubner et al. 2000). Interestingly, all of these characteristics have also been observed in Cx35/36-containing neurones studied in in situ brain slices (Landisman et al. 2002; Galarreta & Hestrin, 1999) and in Cx35/36-positive AII amacrine/ON cone bipolar cells studied in rat retinal slices (Veruki & Hartveit, 2002a,b). Therefore, our data indicate that Cx35/36 gap junctions are likely to mediate rod–rod coupling in the salamander retina. However, as selective gap junctional blockers are discovered, further pharmacological and physiological studies are needed to confirm this claim.
Small gap junctional conductivity mediating salamander rod electrical coupling
Our characterization of rod gap junctional properties revealed a weak coupling of rod photoreceptors. The experimentally measured junctional conductance of 500 pS was much smaller than the previously computer-simulated value of 3.3 nS (Attwell et al. 1984). Yet, it is much closer to the cone–cone coupling conductance found in the mammalian retina (Hornstein et al. 2004; Li & DeVries, 2004). It may be argued that in the slice preparations, the rod network was partially cut off, especially for superficial cell pairs, and that the junctional conductance in vivo might be larger than what we observed here. However, Attwell & Wilson (1980) found that rods were coupled more strongly in the retinal slices than in the flat-mount retina. They speculated that this might be due to the decrease of the number of paths available for current flow away from the site of current injection. Thus the slice preparation itself should not be a significant factor affecting the small rod junctional conductance.
We have quantitatively studied gap junction patterns between rods. We previously calculated the average number of Cx35/36 positive plaques between paired rods to be 4.63 (Zhang & Wu, 2004). Assuming the unitary channel conductance of Cx35/36 is 10–14 pS (Srinivas et al. 1999) and the coupling conductance between paired rods is 500 pS, we estimated that there should be as many as 35–50 channels (500 x 10–12)/(10–14 x 10–12)) that are open under dark-adapted conditions. Each Cx35/36-positive plaque may therefore accommodate 
8–11 activated gap junctions. In a freeze fracture study of gap junctions in the toad retina, Gold & Dowling (1979) showed that the average area per junction in rod–rod pairs was 0.15 ± 0.05 µm2 and the density of junctional particles was 5 x 103µm–2. This suggests that the number of junctional particles in a plaque would be 750 (0.15 x 5 x 103). This also suggests that the activation of 8–11 gap junctions in the dark only represents 1–1.5% (8/750 or 11/750) of the total number of channels in a cluster. This percentage is much smaller than the estimate of 10% of the total number of channels in a cluster assumed to be activated as suggested in a Cx43 study by Bukauskas et al. (2000).
The low coupling conductance (500 pS) resulted in a small coupling coefficient of 0.07, which indicates a weak coupling strength between rods. This weak interconnection between rods may be desirable, because the greatest sensitivity of rods is at the effective operational range, i.e. near the dark membrane potential (Attwell & Wilson, 1980; Capovilla et al. 1987; Yang & Wu, 1996, 1997), and the weak coupling ensures a given signal divided into smaller signals that will fall within the sensitive and linear operational range of rods. These small signals are then expected to be transmitted/amplified precisely and maximally to the second-order cells by means of elaborate chemical synapses. Likewise, in the CNS (including neocortex, cerebellum and thalamus), this similar coupling coefficient has been shown to depolarize neighbouring interneurones to a subthreshold potential, thereby facilitating the synchronized firing of action potentials (reviewed in Galarreta & Hestrin, 2001). Therefore, we think that the small conductivity of rod gap junctions may be necessary to maintain rods in the optimal state for integrating incoming small signals within a narrow operational range.
The significance of the linearity of rod gap junctions
The voltage independence of rod gap junctions within the rod physiological range combined with the frequency independence of rod junctional currents strongly implies that the rod–rod coupling pathway behaves as a linear resistor. This feature may provide a mechanism to prevent rod uncoupling within the rods' dynamic range and also to improve the signal-to-noise ratio within the whole intensity range of light illumination. While voltage would not be the primary modulator of rod gap junctions, it is possible that they would be modulated by neuromodulators and/or intracellular second messengers. In other electrically coupled retinal neurones, such as horizontal and amacrine cells, dopamine, cAMP, and nitric oxide have been found to modulate the gap junctional conductivity of these cells (Lasater, 1987; Hampson et al. 1992; Mills & Massey, 1995; Xin & Bloomfield, 2000). Correspondingly, this may be true in rod electrical coupling. A recent report showed that Cx35 hemi-channels expressed in Xenopus oocytes were modulated by the cAMP-mediated protein kinase A (PKA) pathway (Mitropoulou & Bruzzone, 2003). The consensus sequence sites for PKA phosphorylation have been identified in Cx35 proteins (O'Brien et al. 1996). Furthermore, the dye diffusion coefficient in cells expressing Cx35 was modulated by PKA (O'Brien et al. 2004), and Cx36 associated proteins were found to be phosphorylated by cAMP (Sitaramayya et al. 2003). Therefore, if Cx35/36 is functionally involved in rod electrical coupling, rod electrical synapses are anticipated to be modulated at the cellular and molecular levels. These synapses may also be targeted by second messenger mediated-signalling transduction pathways or by light-dependent signalling pathways. Further pharmacological and physiological studies of the modulation of rod–rod coupling are needed in order to confirm this hypothesis.
The temporal filtering property of the rod network shapes intensity-dependent rod signals
In this study, as shown in Fig. 6, we demonstrated that the low-pass filtering of higher frequency rod signals might be attributed to the RC properties of the rod plasma membrane. This conclusion added a piece of additional evidence to the mechanisms of the band-pass filtering property of the rod network (Detwiler et al. 1978, 1980; Torre & Owen, 1983; Attwell et al. 1984). We found that voltage responses of coupled rods were gradually attenuated as the frequency of current stimuli increased beyond 5 Hz. This indicates that rod electrical synapses favour the transmission of slow potential changes and it agrees with the previous findings of frequency dependence of Cx35/36-containing retinal neurones and brain interneurones (Veruki & Hartveit, 2002a,b; Galarreta & Hestrin, 1999; Nolan et al. 1999; Landisman et al. 2002). One possible explanation for this finding is that Cx35/36 gap junctions might impart a ‘low-pass’ filtering property (Veruki & Hartveit, 2002a,b; Nolan et al. 1999; Galarreta & Hestrin, 2001). However, we found that the transjunctional current under voltage clamp conditions was not significantly altered as the frequency increased (Fig. 5C), indicating that DC coupling is attributed to rod gap junctions. Thus, our data argue that the attenuation of voltage responses of coupled rods by signal frequencies is not the result of the frequency-dependent processes in the gap junctions between rods. Instead it reflects rod membrane RC filtering properties. This argument is supported by our mathematical calculations in which we showed that the cut-off frequency (32 Hz) was very close to the value (33 Hz) measured experimentally, which is a result consistent with the notion that gap junctions in salamander rods behave in a linear manner. A similar observation was also made in the study of primate cone–cone coupling (Hornstein et al. 2004).
Rod photoreceptors are non-spiking neurones in that they respond to light with graded hyperpolarization (Schwartz, 1976; Yang & Wu, 1997). The current response to a single photon is about 2 pA with a rise time of about 2 s (Baylor et al. 1979; Baylor & Nunn, 1986). When light becomes brighter, rod light responses become larger with a faster rise time (Bader et al. 1978; Yang & Wu, 1997). For a saturating light flash, the peak current response is about 50–100 pA and with a rapid rise time of 20–50 ms (Baylor & Nunn, 1986). The frequency-dependent attenuation of the coupled rod network (Fig. 7) allows small signals with slow kinetics to spread to the neighbouring rod without much attenuation, whereas large responses with faster kinetics could be attenuated. This may be a mechanism used by the retina to suppress rod inputs to second-order cells during bright light so that the cone input can take a more dominant role.
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References |
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